JP3935497B2 - Remedy - Google Patents

Description

  The present invention relates to a therapeutic agent that utilizes the phagocytic ability of macrophages to normalize dysfunctional macrophages or is effective against various infectious pathogens. The therapeutic agent (drug or preparation) according to the present invention covers all substances and aggregates given for therapeutic and / or diagnostic purposes, and the preparation includes a drug (in some cases a drug carrier) ) And a drug carrier.

I. Macrophages First, macrophages that play a core function in the action of the therapeutic agent according to the present invention will be outlined.

  The morphology and function of macrophages are described in detail in, for example, Kiyoshi Takahashi et al., “Macrophones that Support Life”, Bunkodo, 2001. The background relating to the present invention will be described as follows. Macrophages are cells that constitute the mononuclear phagocyte system (MPS: Mononuclear Phagocyte System). Monocytes in the blood are derived from hematopoietic stem cells in bone marrow cells, divide and differentiate in the bone marrow, flow into the blood, settle in various tissues, and differentiate into monocyte cells called various names. These cells are histiospheres in connective tissue, Kupffer cells in the liver, alveolar macrophages in the lung, macrophages in lymph nodes / spleen, thorax / peritoneal macrophages in body cavities, osteoclasts in bone, Langerhans cells in skin, small in nerve tissue Glial cells, called microglia in the brain, and type A cells in the synovium, have tissue-specific properties.

1. General properties
(1). Mononuclear cells with a diameter of about 15-20 microns, rich in cytoplasm, and easily adhere to glass and plastic surfaces. Exercises with a false foot and exercises a strong foreign body phagocytosis.
(2). Cells in charge of biological defense.
(3). Has foreign body exclusion function and immunity.
(4). Presenting antigen information to T lymphocytes to establish immunity.
(5). Activated by interferon and becomes an effector of cellular immunity.
(6). X-ray resistant compared to lymphocytes.

2. Physiological significance of macrophages (1). Phagocytosis The most well-known function of macrophages is phagocytosis. This function can be considered as one of the most basic functions that have been available since the era of single cells as life was born and evolved. Therefore, one of the characteristics of macrophages is that their presence is universal across species. This feature appears to provide one of the major advantages of studies targeting macrophage function. In other words, even when research and development of therapeutic agents for mammalian diseases is performed, macrophages other than mammals can be useful both functionally and as research materials. This is due to the fact that macrophages are phylogenetically conserved cells.

(2). Biological defense function The next most important function of macrophages is a bioprotective action. This action is called non-specific bioprotective action, but it is becoming clear from recent studies that the macrophage bioprotective action is also specific. Originally, the term “non-specific” corresponds to the antigen specificity and immune memory that are characteristic of T cells. At present, it has been proved that antigen specificity and immune memory in a strict sense exist in macrophages. It is not a mistake that the macrophage's defenses are nonspecific. However, depending on the type of pathogen, for example, macrophage responses may be qualitatively different, and some of this qualitatively different responses may correspond to differences in receptors that recognize macrophage cell surface pathogens. As shown, the action of macrophages is specific when viewed from the side of cellular responses to foreign (or environmental) stimuli.

  At present, taking into account the above points, the biological defense action centering on macrophages is regarded as the innate immune system, and the biological defense action centering on T cells is regarded as the acquired immune system. Is becoming common. Furthermore, in view of the phylogenetic universality of macrophages, it is natural that the innate immune mechanism is also a biological defense mechanism highly conserved.

(3). Innate immunity mechanism The innate immunity mechanism centering on macrophages is not only in the species that do not have the acquired immune mechanism, but also in the biological species that has the acquired immune mechanism, such as foreign body identification and exclusion mechanism. It plays the core of the mechanism. Even in organisms with acquired immune mechanisms, the identification and elimination of foreign substances such as pathogens is mostly covered by the function of the innate immune mechanism, but when this is insufficient, the acquired immune mechanism is mobilized. Even in this case, antigen presentation by macrophages or the like is essential for specific foreign substance identification, and cells that constitute the innate immune mechanism such as macrophages also play a central role in the elimination mechanism when excluding foreign substances.

(4). Foreign body exclusion On the other hand, endogenous foreign bodies (for example, removal of virus-infected cells) and the like are eliminated by cytotoxic T cells characteristic of acquired immunity, and both proliferation and maturation of the cytotoxic T cells. In addition, another T cell that has received antigen presentation by macrophages or the like is essential. In other words, in order for the acquired immunity to perform a sufficient function, it is assumed that the innate immune mechanism functions completely and purposefully.

(5). Dysfunction Therefore, dysfunctions such as macrophage phagocytosis and antigen presentation can uniquely be a potential cause of immune deficiency. Specifically, the following are known as dysfunctions and diseases related to the points described in I.1 (General properties of macrophages). That is,
1). Leukocyte adhesion deficiency, Chediak-Higashi syndrome and the like are known as phagocytic dysfunctions as abnormal phagocytosis of foreign bodies. In either case, an abnormality is observed in the phagocytic function, and in the latter case, there is an abnormality in the transport of the lysosomal enzyme to the phagocytic cavity, so the bactericidal ability is reduced and the phagocytic ability is remarkably enhanced.
2). The disease as an abnormality of the function in charge of biological defense includes chronic mucocutaneous candidiasis. The macrophages of patients suffering from this disease have reduced migration ability and reduced Candida bactericidal ability.
3). As a disease as a foreign body exclusion function and an abnormality of immunity, Wiscot-Aldrich syndrome can be mentioned. Patient macrophages exhibit complex immune abnormalities such as migration abnormalities and defects in antibody-dependent cytotoxicity.
4). A major histocompatibility (MHC) class II antigen deficiency that causes severe immunodeficiency, although T cells and B cells are healthy, is known as an antigen information presentation function abnormality.
5). Functional abnormality in that it is activated by interferon and becomes an effector of cellular immunity. Infants lacking interferon receptor cannot protect against M. tuberculosis infection, and M. tuberculosis infection becomes lethal. Body deficiency is known.

  Furthermore, mammals deficient in acquired immune cells exist and can survive, but macrophage-deficient animals cannot exist. It is also becoming clear that biologically active substances collectively called cytokines that transmit information between cells perform important functions in biological defense mechanisms centering on foreign substance identification / exclusion. The types of cytokines produced and secreted by macrophages are extremely diverse. As described above, the function of macrophages is essential for maintaining the homeostasis of an individual in terms of foreign substance identification / exclusion.

(6). Anatomical features The anatomical features of macrophages differ depending on the tissue-specific macrophages that have unique characteristics established in various tissues. This is also apparent from the mucosal tissue that is the contact point between the individual and the environment, and specific macrophages are resident in the submucosa of the respiratory tract, digestive tract, and urogenital tract, respectively. These tissue-specific macrophages have a biological response with internal and external environments that are tissue-specific. This suggests that macrophages play an important role in maintaining homeostasis beyond the identification and elimination of foreign substances. The physiological significance of tissue-specific macrophages is largely unknown. On the other hand, from the new viewpoint, the existence significance of the tissue-specific macrophages may be noticed only in relation to various pathological conditions.

(7). Relationship with pathological condition Because the physiological significance that macrophages are located in the core of immune system is strongly suggested that the functional abnormality of tissue-specific macrophages is related to the induction of tissue-specific pathological conditions. is there. In fact, in Crohn's disease, which is one of inflammatory bowel diseases, and intractable diseases such as autoimmune diseases such as rheumatism and age-related diseases such as osteoporosis, macrophage dysfunction is accompanied in some form. Chronic infections of mycobacteria such as Mycobacterium tuberculosis may also be considered to be due to the dysfunction of alveolar macrophages, apart from the problem of acid-fast bacteria themselves. In this way, the development of therapeutic agents that increase the phagocytic ability of macrophages as target cells and consequently increase the concentration of therapeutic agents in macrophages is currently an infectious disease with no effective treatment. It can be said that this is a very important and rational subject in providing new treatments for intractable diseases including

II. Macrophage dysfunction and disease (1). Macrophages as an infectious agent medium According to WHO, tuberculosis, AIDS, malaria, etc. are chronic intractable infections that must be emphasized on a global scale. For example, more than 8 million tuberculosis patients occur each year, and 3 million people die. It is an urgent task to develop a therapeutic drug (drug / preparation) effective for these diseases, and its social significance is extremely great.

  By the way, macrophages are one of the cells that play the most important functions in the living body regarding defense and removal of pathogen infection. In fact, macrophages are distributed throughout the body and organs. These macrophages have different forms and functions depending on the organs and organs present, but they are common in that they protect and eliminate pathogen infection.

  On the other hand, infectious agents have acquired various means to avoid macrophage attack in the course of evolution. In addition, many infectious agents are latent in macrophages and host macrophages. Pathogens that have succeeded in infesting macrophages in this manner can cause chronic and repetitive infections, as well as fatal consequences. That is, in this case, macrophages that should originally achieve protection / removal of pathogen infection turn to function as an infectious pathogen medium.

(2). A typical example of pathogens in macrophages can be seen in tuberculosis. That is, pathogens (Mycobacterium tuberculosis or Mycobacterium bovis) are phagocytosed by alveolar macrophages, the initial infection route, but the phagosomes formed (phagosomes) ) Is stably present in the parentheses. In other words, pathogens can survive as “shelters”, which are macrophages that should otherwise digest them. Other than this, Mycobacterium leprae, the causative agent of rye, Mycobacterium avium, the causative agent of atypical mycobacteria, and Chlamydia pneumonia, the causative agent of chlamydiasis ( Many causative organisms of refractory infectious diseases that have not been established yet, such as Chlamydia pneumoniae, Chlamydia trachomatis, or Chlamydia psittaci, are also infected with macrophages. There is a commonality in becoming a pathogen medium.

  Mycobacterium tuberculosis, AIDS virus, and the like are currently devoted to prevention. However, once infection is established, there is no effective treatment. Even if a compound having a direct bactericidal action against an infectious pathogen is present, in general, a specific pathogen is killed by simply administering the relevant therapeutic agent (in the present invention, killing all pathogens) Or it means killing some and killing it). It is not easy to achieve a concentration in the pathogen-bearing macrophages sufficient to).

  Therefore, by administering an effective therapeutic agent, even though extracellular pathogens can be killed, macrophages as pathogen media still survive as pathogen sources and continue to supply pathogens. The reason why there is currently no radical treatment for pathogens that parasitize in macrophages can be determined from the above points. The present invention aims at this point, and conversely, if the pathogen-infected macrophage as the pathogen medium can be killed or the pathogen in the pathogen-infected macrophage can be killed, many of the above chronic refractory diseases Infectious diseases can be fundamentally treated. For this reason, it is an object of the present invention to provide a therapeutic agent for a disease caused by dysfunction of macrophages or by mediating macrophages.

  As a result of diligent research, the present inventors have killed pathogen-infected macrophages as pathogen media, killed pathogens in pathogen-infected macrophages, or became abnormal due to disease. The present invention has been completed in order to obtain a completely new idea for the purpose of acting on macrophages.

The therapeutic agent of the present invention contains a lipopolysaccharide of Pantoea agglomerans, enhances the phagocytic activity of macrophages, acts on dysfunctional macrophages, and has a cytotoxic effect on lung cancer cells For lung cancer .

Moreover, it is desirable that the macrophages are resident in mucosal tissues. As a result, the disease can be effectively treated at the site where the pathogen causes the initial infection, such as the respiratory tract, digestive tract or genital organs.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2002-247871 which is the basis of the priority of the present application.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the technical scope of the present invention is not limited by these embodiments.

  One of the functions specific to macrophages is phagocytosis, which is provided in all macrophages. The phagocytosis is a function of actively taking up solids having a size of about 1 micron or more into macrophage cells.

  By the way, if an artificially produced solid is actively phagocytosed, it is possible to accumulate the solid in a concentration that cannot normally be achieved in macrophages. Such solids can generally be provided as particles, but in order to actively phagocytose, the particle size advantageous for phagocytosis, the surface characteristics of the particles (having a charge, having a certain flexible structure, etc.), etc. Need to optimize. For example, it is possible to prepare particles that are easily phagocytosed by macrophages using a mixed material of polylactate and polyethylene glycol as a base material.

  Therefore, if a particle is prepared by mixing a drug that acts on an infectious pathogen or a pathogen-infected macrophage, the drug is also actively taken into the macrophage as the particles are phagocytosed.

  The basis of the present invention is a disease (I. 2. (5), (7), II. (1), mycobacteriosis, AIDS, chlamydiasis, toxoplasmosis or cancer). Therefore, the above object is achieved by preparing a preparation containing a drug effective for these in microparticles (drug carrier) that can be phagocytosed by macrophages.

  In general, a preparation that is a combination of a drug carrier and a drug needs to be designed to avoid phagocytosis by macrophages. However, the present invention is novel in that it actively utilizes the phagocytic activity of macrophages based on the idea completely reversed from the conventional one.

  As described above, phagocytosis is one of the defense functions of macrophages. The phagocytosis is a unique function that is characteristically observed in macrophages, and can take up particles of a size that cannot be taken up by cells other than macrophages. Pathogenic microorganisms such as bacteria are engulfed by macrophages and decomposed in macrophages. Therefore, one of the biological significance of macrophage phagocytic function is to damage pathogenic microorganisms. By the way, macrophages are activated by phagocytosis and may be able to counter pathogenic microorganisms. This is a phenomenon known as macrophage activation by phagocytosis, which allows macrophages to injure even cancer cells. Therefore, if macrophages can be activated by phagocytosis and phagocytic activity can be enhanced, pathogenic microorganisms can be more strongly killed.

  In order for particles to be phagocytosed by macrophages, it is considered necessary to have the following properties (phagocytosis).

That is,
-The particle diameter is 1-6 microns. Such particles are called fine particles.
-The particle surface gets wet with macrophage culture solution (in vivo, the body fluid around the macrophage), but does not dissolve immediately but exists as particles for a certain period of time.
-The particles are solid in the temperature range of 20-45 ° C.
-The specific gravity of the particles is greater than the macrophage culture fluid (the body fluid around the macrophages in vivo).
・ The particles also have a surface layer that allows water and ions to penetrate.

  On the other hand, considering that the particles are administered in vivo, the particle surface needs to have a polymer layer with high biocompatibility, and while maintaining the shape of the particles until taken up by macrophages, the macrophages It is necessary to be decomposed into a non-toxic component in the living body and metabolized after it is taken in or into the living body (biodegradable).

  Poly (lactic acid / glycolic acid) copolymer (hereinafter referred to as “PLGA”) or polylactic acid (hereinafter referred to as “PL”) are candidates as a polymer that satisfies the above two conditions and can be easily molded into particles. . PL is more hydrophobic than PLGA and takes a long time to decompose. On the other hand, the decomposition rate of PLGA varies depending on the monomer ratio. The longer the molecular weight, the longer it takes to decompose. When the molecular weight of PLGA is 1,000 or less, it may exist as a liquid in a temperature range of 20 to 45 ° C. Therefore, the molecular weight of PLGA existing as a solid in the temperature range of 20 to 45 ° C. is desirably 1,500 or more.

  Furthermore, the release of the drug contained in the PLGA particles from the inside of the particles is almost zero order in the case of PLGA having a molecular weight of about 20,000. That is, the discharge amount is always kept constant. However, it has been observed that PLGA particles having molecular weights of about 44,000 and 75,000 are not zero-order released but become a pulse type in which the drug is released after a certain time. Moreover, the time during which pulse-type drug release is observed is delayed for PLGA preparations with large molecular weights. That is, the pattern of drug release from PLGA particles depends on the molecular weight of PLGA. Furthermore, the degradation rate of PLGA related to drug release is not only slowed as the molecular weight of PLGA increases, but also in vivo such as in macrophages is predicted to be faster than in vitro, so the molecular weight is up to 150,000. If it is within the range, it is considered that it can be effectively used in vivo.

  From the above viewpoints, a fine particle formulation (acceptable range) having a molecular diameter of 1,500 to 150,000 and a lactic acid / glycolic acid monomer ratio of 50:50 to 75:25 and a particle diameter of 1 to 6 microns, a molecular weight of 5,000 to 75,000, and lactic acid・ Particulate preparations consisting of PLGA with a monomer ratio of glycolic acid of 50:50 to 75:25 and having a particle diameter of 1 to 6 microns (suitable range) are easily phagocytosed by macrophages. The drug is considered to be optimal for the purpose of releasing the drug while maintaining a certain persistence.

Providing a novel therapeutic agent that enables specific killing of macrophages carrying infectious pathogens Actively utilizing the specific phagocytic activity of macrophages carrying various infectious pathogens including Mycobacterium tuberculosis, In order to kill the pathogen in the macrophage or the macrophage itself holding the pathogen, the following therapeutic agents are effective.
(1) .Therapeutic agents that enhance macrophage phagocytic activity
(2). Therapeutic agent with direct killing effect against infectious pathogens in macrophages
(3). Therapeutic drugs that act on macrophages

(1) The therapeutic agent preferably has a property that the macrophages infected pathogens holding / non-holding may be selectively phagocytosed. Conventionally, it has been known that a substance that activates the phagocytic ability of macrophages exists (known matter). However, no attempt has been made to develop therapeutic agents that act on pathogens in macrophages by actively utilizing this property (new matter). The types (2) and (3) are not only applicable to various drugs that act directly on pathogens, but also genes such as DNA or RNA that have the effect of altering the physiological function of pathogen-retaining / non-retaining macrophages. Can be used as No attempt has been made to develop a therapeutic agent effective for removing infectious pathogens combining (1), (2), and (3), and the therapeutic agent of the present invention is effective for treating infectious pathogens. It is a new type of treatment (new matter).

For example, Antimicrobial Agents and Chemotherapy (1998 Vol. 42, No. 10, pp.2682-2689) discloses the antitubercular action of particles containing rifampicin in PLGA. However, this report makes no mention of enhancement of phagocytic activity by PLGA particles. Furthermore, since rifampicin-containing PLGA particles have not improved the efficacy as an antituberculosis drug compared to single rifampicin, it is clear that the rifampicin-containing PLGA particles have different properties from the particles of the present invention. Furthermore, as shown in FIGS. 4 and 5, the molecular weight of PLGA is important for controlling the release of rifampicin. However, the above report does not describe the molecular weight of PLGA used in the preparation of PLGA. Presumably, PLGA having a molecular weight different from that of the particles of the present invention was used, so that it did not have a function as an antituberculosis drug. When preparing PLGA particles with antituberculous drug activity, use the appropriate PLGA molecular weight, monomer ratio, and diameter of the prepared particles, and evaluate the uptake of particles by macrophage phagocytosis and antituberculosis activity in macrophages I have to do it. To sum up the above points, the PLGA particles described in the above paper have a certain commonality in terms of materials, such as the PLGA monomer ratio and particle diameter are within the “suitable range” of the present invention. It does not describe the exact properties of the particles and has no antituberculosis activity. Therefore, it is difficult to be a precedent that denies the novelty and inventive step of the present invention.

Furthermore, a method for preparing rifampicin-containing particles prepared using PLGA with a molecular weight of 82,500 is disclosed in Pharmaceutical Research 2000 (Vol. 17, No. 8, pp.955-961). The method is different from the present invention. The effect of rifampicin-containing PLGA particles using PLGA with a molecular weight of 82,500 is disclosed in Pharmaceutical Research (Pharmaceutical Research 2001, Vol. 18, No. 9, pp. 1315-1319). In view of this, the antituberculosis activity of rifampicin-containing PLGA particles does not reach the effect of rifampicin alone in vitro, and it cannot be said that there is an obvious therapeutic effect in animal experiments. In order for rifampicin-containing PLGA particles to exhibit an antitubercular effect, the particles must have moderate degradability and a high rifampicin inclusion rate. However, in the above-mentioned paper, anti-tuberculosis activity has been investigated without examining the properties that influence such activity. It is known that the degradability and the encapsulation rate decrease in PLGA particles having a large molecular weight, so it seems that the use of PLGA having a high molecular weight of 82,500 did not exhibit the antituberculosis effect. As described above, research similar to that presented in the present invention has been conducted so far, but as in the present invention, it is intended to use the phagocytic activity of macrophages and achieve this activation. Thus, no attempt has been made to kill intracellular parasitic pathogens. In addition, although rifampicin-containing PLGA has been prepared, no appropriate research has been conducted on preparation methods and material properties for the purpose of achieving the object of the present invention. There is a slight description in Pharmaceutical Research (Pharmaceutical Research 2001, Vol. 18, No. 10, pp.1405-1410) that rifampicin-containing PLGA particles are assumed to be taken up by macrophages. However, in this case as well, a preparation intended to be actively incorporated into or activated by macrophages has not been prepared, and the phagocytosis rate of rifampicin-containing PLGA particles has not been studied. Incidentally, the concentration of rifampicin in macrophages in vitro using the rifampicin-containing PLGA particles described in this report is only 0.45 μg / 10 ⁶ cells. When the rifampicin-containing PLGA microparticles according to the present invention are used, not only phagocytosis is activated, but the concentration of rifampicin in macrophages reaches ⁶ μg / 10 ⁶ cells, which is 13 times or more. From the above, it is clear that if phagocytosis is induced in macrophages and the pathogenic microorganisms in the macrophages are intended to be killed, it is difficult to achieve this by simply containing a drug in the microspheres. Moreover, in any report so far, there has been no report examining whether rifampicin-containing PLGA particles kill intracellular tuberculosis using alveolar macrophages which are target cells of M. tuberculosis. As is clear from the above-mentioned “macrophages that support life”, macrophages are highly tissue-specific and results obtained using macrophages present in blood are immediately compared to tissue-specific macrophages such as alveolar macrophages. It is a well-known fact that neither can be applied. That is, the novelty of the present invention is not limited to the novelty of the concept, but as an example supporting this concept, the alveolar macrophage is used, and the phagocytic activity of the cell is induced, and the alveolar macrophage actually The high drug concentration is maintained, and this preparation actually produces and provides rifampicin-containing PLGA particles that are clearly superior in killing M. tuberculosis in alveolar macrophages compared to rifapicin alone.

  On the other hand, it is already widely known that the production of non-specific antibacterial substances such as hydrogen peroxide and oxygen radicals is enhanced from macrophages by phagocytosing particles to macrophages. For example, in the European Journal of Pharmaceutical Sciences 2000 (Vol. 15, pp.197-207), by phagocytosing PLGA particles, blood monocytes are peroxidized from cultured macrophages of similar character. It is disclosed that hydrogen production is enhanced. However, it has not been described that phagocytic activity is enhanced when macrophages phagocytose PLGA. Furthermore, since the activation of macrophages is extremely diverse, an increase in hydrogen peroxide production by phagocytosis is not directly linked to enhanced phagocytosis.

In the actual treatment of infectious diseases, a preparation in which a drug of type (2) or (3) is contained in a therapeutic drug of type (1) is effective. In other words, in order for the therapeutic agents of types (2) and (3) to act effectively in macrophages, the therapeutic agent of (1) enhances the phagocytic activity of macrophages (2) and (3) types. Enhances the function of transporting therapeutic agents into macrophages. That is, as shown in FIG. 1, the therapeutic agent that is the subject of the present invention is easily phagocytosed by macrophages and enhances the phagocytic activity of macrophages when phagocytosed. The concentration of the therapeutic agent is significantly higher. That is, the drug-encapsulated microparticles according to the present invention on the left side are positively taken into the macrophages and the drug concentration in the macrophages becomes significantly higher than the drugs taken into the macrophages in the conventional drug solution on the right side. Thus, “increasing the phagocytic activity of macrophages” described in the claims means that the therapeutic agent increases its phagocytic ability, so that the treatment within the macrophage is greater than when the therapeutic agent is simply administered. This means that the concentration of the drug will be significantly higher.

Specific Application Example: Tuberculosis Tuberculosis is described as an example of the effectiveness of the therapeutic agent presented in the present invention. Mycobacterium tuberculosis enters the alveoli from the airways by droplets and is phagocytosed by alveolar macrophages. Normally, phagocytosed pathogens are destined to be broken down by proteolytic enzymes in the cell. However, Mycobacterium tuberculosis avoids proteolytic enzyme attack and survives in macrophages. The Mycobacterium tuberculosis in the macrophage moves outside the macrophage and continuously supplies the Mycobacterium tuberculosis into the host body. Currently, drugs such as isoniazid, rifampicin, streptomycin sulfate and ethambutal are used as anti-tuberculosis drugs. Both drugs are effective against Mycobacterium tuberculosis outside macrophages, but have no effect on Mycobacterium tuberculosis within alveolar macrophages. This is mainly due to the lack of sufficient drug concentration in the alveolar macrophages to kill M. tuberculosis.

  Therefore, if the drug concentration sufficient to kill M. tuberculosis is obtained in the alveolar macrophages using the phagocytosis of alveolar macrophages, M. tuberculosis in alveolar macrophages can also be killed. In this case, phagocytosis is used to selectively increase the drug concentration in macrophages.

A. Therapeutic agent that enhances phagocytic ability of macrophages Taking PLGA as an example, a method for producing a therapeutic agent that enhances phagocytic ability of macrophages and the effects thereof will be described.
I. Enhancement of macrophage phagocytosis by PLGA microparticle formulation (PLGA microparticles themselves are medicinal ingredients in this section)
1. PLGA fine particle preparation method
(a) Material
(1) .PLGA (poly (lactic acid / glycolic acid) copolymer) monomer ratio: 75:25, molecular weight 20,000 (Wako Pure Chemical Industries, Ltd .: PLGA-7520)
(2). PVA (polyvinyl alcohol) polymerization degree 500

(b) Preparation of PLGA fine particle formulation
(1). Dissolve 500 mg of PLGA in 1.5 mL of methylene chloride.
(2) Dissolve PVA in water to 0.3% (w / v).
(3) When 8 mL of the PVA aqueous solution of (2) is added to the solution of (1) and stirred for 3 minutes, an oil-in-water (o / w type) emulsion is formed.
(4) Add (3) to 200 mL of the PVA aqueous solution of (2), and stir at 520 rpm for 3 hours at room temperature.
(5) The microparticle preparation is settled and separated by centrifugation (3,000 rpm / 15 minutes), and 10 mL of distilled water is further added and washed twice by centrifugation.
(6) .1 Drying under reduced pressure in a desiccator day and night.
(7) The particle size distribution of the obtained fine particle formulation is shown in FIG. As is apparent from this figure, the prepared microparticle formulation has a peak at a diameter of about 2 microns and a distribution between 1 and 10 microns. These particles are solid at room temperature. The yield calculated from the total weight of PLGA (500 mg) used for preparation and the recovered preparation was about 90%.
(8). Other Examples The properties of PLGA fine particles (molecular weight and composition) prepared are summarized below.
1. PLGA-5005 (PLGA molecular weight 5,000, lactic acid / glycolic acid 50:50)
2. PLGA-5010 (PLGA molecular weight 10,000, lactic acid / glycolic acid 50:50)
3. PLGA-5020 (PLGA molecular weight 20,000, lactic acid / glycolic acid 50:50)
4. PLGA-7505 (PLGA molecular weight 5,000, lactic acid / glycolic acid 75:25)
5. PLGA-7510 (PLGA molecular weight 10,000, lactic acid / glycolic acid 75:25)
The PLGA fine particle preparation method is the same as 1. (b) (1) to (6) except for the type of PLGA.

  The average particle size of the obtained fine particle preparation was about 2 microns regardless of which PLGA was used. The yield calculated from the total weight of PLGA (500 mg) used for the preparation and the recovered preparation was about 90%.

2. Enhancing phagocytosis of alveolar macrophages by phagocytosing PLGA microparticles
(1). Prepare alveolar macrophage cells (NR8383 cells) at 1 × 10 ⁶ cells / mL in medium (Ham F-12K, 15% fetal calf serum), add to 24-well plate, and add PLGA-7520 Add 0.04, 0.4, and 4 micrograms (μg) of the microparticle formulation, and culture in a carbon dioxide incubator (37 ° C).
(2) After culturing for 1 hour, remove the culture solution, add 0.1 mL of physiological saline (PBS) containing 0.25% trypsin / phosphate buffer, leave it at room temperature for 5 minutes, remove the culture supernatant, Wash.
(3) Add 0.1 mL of medium to this and mix with 0.1 mL of 80% Percoll (Pharmacia) to prepare a 40% Percoll solution, and add this to 0.1 mL of 70% Percoll in a 1.5 mL sample tube. Overlay and centrifuge (8,000 rpm for 10 minutes).
(4) After centrifugation, the cells present at the interface were collected, the cells were washed with PBS, 1 mL of medium was added, and polystyrene latex particles (FITC-) labeled with fluorescein isothiocyanate (FITC) having a particle size of 2.0 microns. Add 1 × 10 ⁷ PSLP) and incubate for 1 hour. The reason for labeling with fluorescent FITC is to facilitate quantification.
(5) After culture, centrifuge (700 rpm / min, 5 min), add 1 mL of PBS to the macrophages in the sedimentation layer, and repeat the centrifugation twice.
(6) After removing PBS, add 0.1 mL of 10% formalin / PBS to the cells, fix for 5 minutes, add 1 mL of distilled water, remove the supernatant, add 1 mL of distilled water again, and remove the supernatant . After removing the supernatant, add 0.1 mL of distilled water to suspend the cells, take 0.02 mL of the suspension, spread on a slide glass, and dry.
(7). Take multiple fields of view under a fluorescence microscope and measure the number of NR8383 cells that have incorporated FITC-PSLP per 100 cells.
(8) Table 1 shows changes in macrophage FITC-PSLP phagocytosis by PLGA microparticle preparations. The effect of the PLGA microparticle preparation on the phagocytic ability of macrophages is not remarkable when added in a low amount, but it is clear that the addition of 0.4 (μg / mL) significantly activates the phagocytic ability.

II. Enhancement of macrophage phagocytosis by lipopolysaccharide Lipopolysaccharide preparation method
(a) Material
(1) .Panthoea agglomerans belonging to the genus Pantoea of Gram-negative bacteria

(b) Preparation of lipopolysaccharide
(1) Pantoea agglomerans was added to 7 liter (L) of broth medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 1 g / L, pH 7.5), and 35 ° C. Incubate with shaking overnight to collect about 70 g of wet cells.
(2) About 70 g of cells were suspended in 500 mL of distilled water, 500 mL of 90% hot phenol was added, and the mixture was stirred at 65 to 70 ° C. for 20 minutes, cooled, and the aqueous layer was recovered. The recovered aqueous layer is dialyzed overnight to remove phenol, and the dialyzed solution is ultrafiltered under nitrogen gas at 2 atm with a molecular weight of 200,000 cut-off membrane.
(3). The obtained lyophilized crude lipopolysaccharide was dissolved in distilled water, and subjected to anion exchange chromatography (Pharmacia, Q-Sepharose Fast Flow). 10 mM Tris-HCl (pH 7.5) and 10 mM The sample solution is passed through the column with a buffer solution containing NaCl, and the Limulus active fraction is eluted with 200 to 400 mM NaCl / 10 mM Tris-HCl (pH 7.5). The eluate is subjected to ultrafiltration under the same conditions as described above, desalted and concentrated, and lyophilized to obtain about 300 mg of purified lipopolysaccharide from about 70 g of wet cells.

2. Enhancement of phagocytosis of alveolar macrophages by lipopolysaccharide
(1). Prepare alveolar macrophage cells (NR8383) at 1 × 10 ⁶ cells / mL in medium (Ham F-12K, 15% fetal bovine serum), add to 24-well plate, and add 1 μg of lipopolysaccharide. / mL, and incubate in a carbon dioxide incubator (37 ° C).
(2) After culturing for 1 hour, transfer the cells to a 1.5 mL sample tube, remove the culture solution by centrifugation (2,000 rpm / min, 5 min), add 0.1 mL of 0.25% trypsin / PBS, and add 5 mL at room temperature. After standing for 1 min, centrifuge (2,000 rpm / min, 5 min) to remove the culture supernatant and wash with PBS.
(3) Add 0.1 mL of medium to this and mix with 0.1 mL of 60% Percoll to prepare a 30% Percoll solution, and centrifuge (8,000 rpm for 10 minutes).
(4) After centrifugation, the cells present on the surface of the solution were collected, the cells were washed twice with PBS, 1 mL of medium was added, and 1 × 10 ⁷ FITC-PSLP with a particle size of 2.0 microns was added, Incubate for 1 hour.
(5) After the culture, remove the supernatant, add 0.1 mL of 0.25% trypsin / PBS and leave it at room temperature for 5 minutes, add 1 mL of the medium, and centrifuge (700 rpm / minute, 5 minutes).
(6). Add 1 mL of PBS to the macrophages in the sedimentation layer, centrifuge (700 rpm / min, 5 minutes), remove the supernatant, add 1 mL of PBS again, and then centrifuge (700 rpm / min, 5 minutes). Remove the supernatant.
(7) After removing the supernatant, add 0.1 mL of 10% formalin / PBS to the cells, fix for 5 minutes, add 1 mL of distilled water, remove the supernatant, add 1 mL of distilled water again, and remove the supernatant. except.
(8) After removing the supernatant, add 0.1 mL of distilled water to suspend the cells. Take 0.02 mL of the suspension, spread on a slide glass, and dry.
(9). Take multiple fields of view under a fluorescence microscope and measure the number of NR8383 cells that have incorporated FITC-PSLP per 500 cells.
(10) The increase in FITC-PSLP phagocytosis of macrophages by lipopolysaccharide is as shown in Table 2. It is clear that macrophage phagocytic activity was activated by lipopolysaccharide.

B. Therapeutic agents that act on pathogens in macrophages
Effective transfer of rifampicin (antituberculosis drug) into macrophages by phagocytosis of RFP-PLGA microparticles

1. Preparation of PLGA microparticle formulation containing rifampicin
(a) Material
(1) .PLGA (poly (lactic acid / glycolic acid) copolymer) monomer ratio: 75:25 or 50:50, molecular weight 5,000, 10,000 or 20,000, Wako Pure Chemical Industries, Ltd .: PLGA-5005, 5010, 5020, 7505 ,
7510, 7520
(2) Rifampicin
(3). PVA (polyvinyl alcohol) polymerization degree 500

(b) Preparation of RFP-PLGA fine particle formulation
(1). Dissolve 500 mg of PLGA and rifampicin (0, 50, 100, 200 mg) in 1.5 mL of methylene chloride.
(2) Dissolve PVA in water to 0.3% (w / v).
(3) When 8 mL of the PVA aqueous solution of (2) is added to the solution of (1) and stirred for 3 minutes, an o / w type emulsion is formed.
(4) Add (3) to 200 mL of the PVA aqueous solution of (2), and stir at 520 rpm for 3 hours at room temperature.
(5) The microparticle preparation is settled and separated by centrifugation (3,000 rpm / 15 minutes), and 10 mL of distilled water is further added and washed twice by centrifugation.
(6) .1 Drying under reduced pressure in a desiccator day and night.
(7). The prepared RFP-PLGA particles (molecular weight and composition) are summarized below.
1. RFP-PLGA5005 (PLGA molecular weight 5,000, lactic acid / glycolic acid 50:50)
2. RFP-PLGA5010 (PLGA molecular weight 10,000, lactic acid / glycolic acid 50:50)
3. RFP-PLGA5020 (PLGA molecular weight 20,000, lactic acid / glycolic acid 50:50)
4. RFP-PLGA7505 (PLGA molecular weight 5,000, lactic acid / glycolic acid 75:25)
5. RFP-PLGA7510 (PLGA molecular weight 10,000, lactic acid / glycolic acid 75:25)
6. RFP-PLGA7520 (PLGA molecular weight 20,000, lactic acid / glycolic acid 75:25)
The particle size distribution and properties of the obtained fine particle preparation were all the same as those of the PLGA-only fine particle preparation (AI 1. (b) (7)).
(8) After dissolving 4 mg of a fine particle preparation containing 0, 50, 100, 200 mg of rifampicin in 500 mg of PLGA in 1 mL of methylene chloride, the absorbance at 475 nm is measured with a spectrophotometer.
(9) Table 3 shows the amount of rifampicin recovered in the RFP-PLGA fine particle formulation (PLGA molecular weight 20,000, fine particle PLGA-7520 composed of lactic acid / glycolic acid 75:25). As is apparent from this result, it can be seen that rifampicin is efficiently formulated.

2. Other RFP-PLGA fine particle formulation preparation methods (membrane emulsification method, publicly known)
RFP-PLGA7510 fine particle formulation was prepared using membrane emulsification method.
The membrane emulsification method is an emulsification method in which one of two kinds of liquids not mixed (dispersed phase) is pressurized and dispersed in the other liquid (continuous phase) through a porous glass film (SPG film). When this method is used, an emulsion having a uniform particle size can be obtained.

(a) .Material
(1) .PLGA (poly (lactic acid / glycolic acid) copolymer) monomer ratio: 75:25, molecular weight 10,000, Wako Pure Chemical Industries, Ltd .: PLGA7510
(2) Rifampicin
(3). PVA (polyvinyl alcohol) polymerization degree 500

(b) Preparation of RFP-PLGA fine particle formulation
(1). Dissolve 500 mg of PLGA and 100 mg of rifampicin in 10 mL of methylene chloride.
(2) Dissolve PVA in water to 0.3% (w / v).
(3) When the solution of (1) is dispersed in 100 mL of the PVA aqueous solution of (2) through an SPG membrane (Ise Chemical Industries pore size 0.49 μm), an oil-in-water (o / w) emulsion is formed.
(4) Add (3) to 200 mL of the PVA aqueous solution of (2), and stir at 520 rpm for 3 hours at room temperature.
(5) The fine particle preparation is settled and separated by centrifugation (3,000 rpm / 15 minutes), and 10 mL of distilled water is further added and washed twice by centrifugation.
(6) .1 Drying under reduced pressure in a desiccator day and night.
(7) The average particle size of the obtained fine particle preparation is 1.98 microns. The PLGA yield calculated from the total weight of PLGA (500 mg) used for the preparation and the recovered preparation was about 90%. The yield of rifampicin was about 75%. These particles are solid at room temperature.

3. Other examples by membrane emulsification method RFP-PLGA fine particles (molecular weight and composition) other than the RFP-PLGA7510 fine particle formulation prepared by using the membrane emulsification method are summarized below.
1. RFP-PLGA5005 (PLGA molecular weight 5,000, lactic acid / glycolic acid 50:50)
2. RFP-PLGA5010 (PLGA molecular weight 10,000, lactic acid / glycolic acid 50:50)
3. RFP-PLGA5020 (PLGA molecular weight 20,000, lactic acid / glycolic acid 50:50)
4. RFP-PLGA7505 (PLGA molecular weight 5,000, lactic acid / glycolic acid 75:25)
5. RFP-PLGA7520 (PLGA molecular weight 20,000, lactic acid / glycolic acid 75:25)
These RFP-PLGA fine particle formulation preparation methods are the same as the membrane emulsification method except for the type of PLGA.

  The average particle size of the resulting microparticle formulation is 2.20 microns for PLGA5005, 2.66 microns for PLGA5010, 2.29 microns for PLGA5020, 2.00 microns for PLGA7505, and 1.85 microns for PLGA7520. The PLGA yield calculated from the total weight of PLGA (500 mg) used for preparation and the recovered preparation was about 90%. The yield of rifampicin was about 87% for PLGA5005, about 78% for PLGA5010, about 67% for PLGA5020, about 91% for PLGA7505, and about 58% for PLGA7520.

4). Release of rifampicin from RFP-PLGA microparticles
(1) 50 mg of each RFP-PLGA fine particle prepared by the membrane emulsification method was dispersed in 5 mL of pH 7.4 phosphate buffer (ionic strength 0.154 M) maintained at 37 ° C. (temperature).
(2) The supernatant was collected by centrifugation over time, and 5 mL of pH 7.4 phosphate buffer (ionic strength 0.154 M) was added to the remaining fine particles.
(3) The concentration of rifampicin eluted in the supernatant was measured. The measurement was carried out using a spectrophotometer at a wavelength of 475 nm.
(4) The ratio of rifampicin released from each RFP-PLGA microparticle into the supernatant is shown in FIG. 4 and FIG. The experimental data showed that the release rate of rifampicin was fast from PLGA5005, PLGA5010, PLGA7505, and PLGA7510 with PLGA molecular weights of 5,000 and 10,000. From these results, it was shown that the composition of PLGA has a molecular weight of 5,000 to 10,000 and a ratio of lactic acid to glycolic acid of 50:50 or 75:25 is excellent in the transfer of drugs via phagocytosis to macrophages.

5). Selective increase in intracellular rifampicin concentration by phagocytosis of RFP-PLGA microparticle formulation (PLGA-7520)
(1) The prepared RFP-PLGA microparticle preparation is redispersed in PBS, centrifuged (400 rpm / min, 5 minutes) to remove large particles, and the size is about 1 to 3 microns by microscopic observation. The microparticle preparation (about 30% by weight) prepared in the above is used in the subsequent experiments.
(2) NR8383 cells cultured in a medium at 5 × 10 ⁵ cells / 0.9 mL are placed in a 24-well plate, to which 0.12 mg / 0.1 mL of each RFP-PLGA microparticle preparation is added, and cultured for 12 hours.
(3) After culture, remove the culture solution, add 0.1 mL of 0.25% trypsin / PBS, leave it at room temperature for 5 minutes, remove the culture supernatant, and wash.
(4). Add 0.1 mL of medium and mix with 0.1 mL of 80% Percoll to make a 40% Percoll solution. This is overlaid on 0.1 mL of 70% Percoll in a 1.5 mL sample tube and centrifuged (8,000 Rotate / minute, 10 minutes).
(5) After centrifugation, the cells present at the interface are collected, the cells are washed with PBS, and rifampicin incorporated into the cells is extracted with methylene chloride.
(6) The amount of rifampicin is determined from the absorbance at 475 nm.
(7). As a control, the same amount of rifampicin in dimethyl sulfoxide (DMSO) contained in 0.12 mg of each RFP-PLGA microparticle preparation was prepared, and this was added to a 24-well plate to which 1 mL of medium was added, and NR8383 cells were added. Add.
(8) After culturing the cells for 12 hours, the cells (5 × 10 ⁵ ) are collected, and rifampicin in the cells is extracted with methylene chloride.
(9) FIG. 3 shows the amount of rifampicin taken up by macrophages. When the amount of rifampicin added is 40 (μg / 5 × 10 ⁵ cells / well / mL), the RFP-PLGA microparticle preparation of this example has about 19 times as much rifampicin as compared with the conventional medium containing rifampicin. It is shown that it is capturing.

C. Drugs that enhance macrophage phagocytosis and act on pathogens in macrophages (A + B)
Enhancement of macrophage phagocytic activity by phagocytosis of RFP-PLGA microparticle formulation 1. Preparation of RFP-PLGA microparticle formulation
(a) Material
(1). (1) PLGA (poly (lactic acid / glycolic acid) copolymer) monomer ratio: 75:25 or 50:50, molecular weight 5,000, 10,000 or 20,000, Wako Pure Chemical Industries, Ltd .: PLGA-5005, 5010, 5020, 7505,
7510, 7520
(2) Rifampicin
(3). PVA (polyvinyl alcohol) polymerization degree 500

(b) Preparation of RFP-PLGA fine particle formulation
(1). Dissolve 500 mg of PLGA and 100 mg of rifampicin in 1.5 mL of methylene chloride.
(2) Dissolve PVA in water to 0.3% (w / v).
(3) When 8 mL of the PVA aqueous solution of (2) is added to the solution of (1) and stirred for 3 minutes, an o / w type emulsion is formed.
(4) Add (3) to 200 mL of the PVA aqueous solution of (2), and stir at 520 rpm for 3 hours at room temperature.
(5) The fine particle preparation is settled and separated by centrifugation (3,000 rpm / 15 minutes), and 10 mL of distilled water is further added and washed twice by centrifugation.
(6) .1 Vacuum drying in a desiccator day and night.
(7) The particle size distribution and properties of the obtained microparticle preparation were the same as those of the PLGA-only microparticle preparation (AI 1. (a) (7)).
(8) Re-disperse the microparticle formulation in PBS, remove the large microparticle formulation by centrifugation (400 rpm / min, 5 minutes), and remove the microparticle formulation adjusted to a size of about 1 to 3 microns by microscopic observation. Used for experiments.

2. Enhancement of macrophage phagocytic activity by phagocytosis of RFP-PLGA microparticles (PLGA-7520)
(1). Prepare alveolar macrophage cells (NR8383) at 1 × 10 ⁶ cells / mL in medium (Ham F-12K, 15% fetal calf serum) and add to 24-well plate. Add 0.012, 0.12, and 1.2 μg, and incubate in a carbon dioxide incubator (37 ° C).
(2). After culturing for 1 hour, transfer the cells to a 1.5 mL sample tube, remove the culture solution, add 0.1 mL of 0.25% trypsin / PBS, leave it at room temperature for 5 minutes, remove the culture supernatant, and wash. Do.
(3) Add 0.1 mL of medium and 0.1 mL of 90% Percoll to this to bring the Percoll concentration to 45%, and then centrifuge (8,000 rpm for 10 minutes).
(4) After centrifugation, cells on the surface of the solution are collected, washed twice with PBS, 1 mL of the same medium used in (1) is added, and FITC-PSLP with a particle size of 2.0 microns is added. Add 1 x 10 ^{7 and} incubate for 1 hour.
(5) After culture, the supernatant is removed, 0.1 mL of 0.25% trypsin / PBS is added and left at room temperature for 5 minutes, and then 1 mL of the medium is added and centrifuged (700 rpm / minute, 5 minutes).
(6). Add 1 mL of PBS to the macrophages in the sedimentation layer, centrifuge (700 rpm / min, 5 minutes), remove the supernatant, add 1 mL of PBS again, and then centrifuge (700 rpm / min, 5 minutes). Remove the supernatant.
(7) After removing the supernatant, add 0.1 mL of 10% formalin / PBS to the cells, fix for 5 minutes, add 1 mL of distilled water, and centrifuge (700 rpm / minute, 5 minutes). except for. To this, 0.1 mL of distilled water is added to suspend cells, and 0.02 mL of the suspension is taken, spread on a slide glass, and dried.
(8). Take multiple fields of view under a fluorescence microscope and measure the number of macrophages (NR8383 cells) that have taken in FITC-PSLP per 500 cells.
(9). Increase in macrophage FITC-PSLP phagocytosis by phagocytosis of RFP-PLGA microparticle formulation is shown in Table 4. It is clear that macrophage phagocytosis was activated by the RFP-PLGA microparticle formulation.

From the above results, it was revealed that (1) PLGA fine particle preparation and lipopolysaccharide activate the phagocytic ability of macrophages. In addition, (2) the transfer of rifampicin into macrophages is greatly increased by inclusion in the PLGA microparticle formulation, and (3) macrophage phagocytic ability even if rifampicin is contained in the PLGA microparticle formulation It became clear that it was activated. Therefore, the rifampicin-containing PLGA microparticle formulation activates the phagocytic ability of macrophages, and as a result, the concentration of rifampicin in the macrophages increases, and the object of the present invention is to effectively act on pathogens retained in the macrophages. Can be achieved. The above results show an example of a novel therapeutic agent that effectively incorporates a therapeutic agent that acts on a pathogen in macrophages by “ enhancing macrophage phagocytic activity”, which is the basic concept of the present invention. is there.

3. Efficacy of phagocytosis of RFP-PLGA microparticles in the macrophage
(a) Method for measuring the survival rate of Mycobacterium tuberculosis (BCG) in macrophages
(1). Dry BCG vaccine was dissolved in physiological saline (12 mg / mL) and stirred, then KRD medium (3-4 mL) was transferred to a T-25 culture flask, 40 μL of BCG suspension was added, and a dry incubator at 37 ° C. Incubated with
(2). In the experiment, the bacterial solution and glass beads were put into a sample tube at a ratio of 1: 4, stirred for 1 minute with a vortex mixer, and further subjected to ultrasonic treatment for 5 minutes with an ultrasonic washing machine. Dispersion was performed.
(3) NR8383 was placed in a 6-well plate at a concentration of 1 × 10 ⁶ cells / mL (total volume 5 mL). Mycobacterium tuberculosis (BCG) was added to the cell culture medium at 10 cells (multiplicity of infection (MOI) = 10).
(4) After infection with a carbon dioxide incubator at 37 ° C for 4 hours, the supernatant was removed after centrifugation at 2,000 rpm for 5 minutes (SCT15B). Using a serum-free medium, the centrifugation was repeated twice to remove bacteria outside the cells.
(5) NR8383 was seeded in a 24-well plate at a concentration of 1 × 10 ⁶ cells / mL.
(6) Each RFP-PLGA fine particle was added to NR8383 at a rate of 10 per cell.
(7) After phagocytosing in a carbon dioxide incubator at 37 ° C. for 4 hours, extracellular particles were excluded using 0.25% trypsin.
(8). 80% percoll was added to make 40% percoll-cell solution, and 70% percoll was added to create a density gradient. After centrifugation at 10,000 rpm for 5 minutes (SORVALL Biofuge fresco), the cells at the interface between 40% and 70% percoll were recovered, and the cells and RFP-PLGA were separated.
(9) Similarly, centrifugation was repeated twice using PBS to remove percoll.
(10) The ratio of viable and dead bacteria was measured using a fluorescein diacetate (FDA /, Fluorescein diacetate) / ethidium bromide (EB) staining method. FDA was dissolved in acetone at a concentration of 5 mg / mL, and 20 μL was diluted with 1 mL of PBS at the time of use. EB was dissolved in PBS at a concentration of 20 μg / mL, and 50 μL was diluted with 1 mL PBS before use. For staining, equal amounts of diluted FDA and EB were mixed and used. This mixed solution was placed on a 1 μL slide glass, and 1 μL of the bacterial solution was placed thereon, placed at room temperature for 2 minutes, and observed with a fluorescence microscope. In viable bacteria, FDA is decomposed by the esterase activity derived from the bacteria, and emits green fluorescence. On the other hand, since dead cells have no esterase activity, staining with FDA is not observed, but they are stained with EB and emit orange fluorescence.

(b) Efficacy of RFP-PLGA microparticles incorporated into macrophages by phagocytosis
(1). In order to investigate the effect of RFP-PLGA microparticles on the killing of M. tuberculosis in macrophages, RFP-PLGA microparticles were administered to macrophages that had previously been phagocytosed by M. tuberculosis (M. tuberculosis-infected macrophages), and these microparticles were phagocytosed by macrophages. I let you.
(2). FIG. 6 shows the results of investigating what kind of killing effect RFP-PLGA migrated into macrophages by phagocytosis against M. tuberculosis in macrophages. Even RFF-PLGA microparticle doses containing rifampicin about 20 times less than rifampicin alone (100 μg / mL) (MOI = 10, estimated rifampicin amount is 5 μg / mL, so rifampicin in the microparticle formulation is rifampicin alone It is clear that the intracellular concentration of rifampicin is significantly higher. Administration of RFP-PLGA microparticles to Mycobacterium tuberculosis-infected alveolar macrophages killed M. tuberculosis intracellularly more than 20 times more efficiently than rifampicin administered alone. In other words, by administering the drug via phagocytosis of particles, the therapeutic index was improved by 20 times or more regardless of the antitubercular action of the drug itself.

Providing therapeutic agents that act on macrophages that are in a dysfunctional state by enhancing the phagocytic activity of macrophages It is known that many macrophages are usually infiltrated into cancer tissue. This is based on the accumulation of macrophages for the purpose of eliminating cancer cells because cancer cells are foreign to the living body. If the macrophage function is normal, the cancer cells are damaged and disappear, but if the macrophage function is abnormal, the cancer cells cannot be damaged and the cancer cells grow and form a tumor mass. By the way, as already described, macrophage activation can damage the cancer cells. Accordingly, it is possible to effectively treat cancer by causing the macrophages that have become functionally abnormal by enhancing the phagocytic activity of macrophages to acquire a cancer cytotoxic effect.

  On the other hand, as shown in Table 2, phagocytosis of alveolar macrophages was enhanced by 166% by treatment with lipopolysaccharide (1 μg / mL), and alveolar macrophages were activated by lipopolysaccharide. Thus, alveolar macrophages whose phagocytic ability is activated by lipopolysaccharide are considered to exert cytotoxic effects on lung cancer cells. Therefore, here, an example of lung cancer cytotoxicity of activated alveolar macrophages by lipopolysaccharide is shown.

Specific application example: Lung cancer The lung cancer cytotoxic effect by activating alveolar macrophages is shown as an application example.
1. Activation of alveolar macrophages by lipopolysaccharide and co-culture of alveolar macrophages and Sato lung cancer cells
(1) NR8383 cells were placed in 24-well plates at a concentration of 1 × 10 ⁶ / mL (total volume 1.5 mL). As controls, 150% of 5% fetal bovine serum, 150 μL of F-12K medium, and 10 μg / mL lipopolysaccharide were added.
(2) It was left in a carbon dioxide incubator at 37 ° C for 24 hours.
(3) Sato lung cancer cells were prepared to a concentration of 1 × 10 ⁵ cells / mL, and 50 μL per well was added to a 96-well plate.
(4). The NR8383 treated with the section (1), adjusted to a concentration of ^{5 × 10 5, 1 × 10} 5, 5 × 10 4, 1 × 10 4 cells / mL, and placed 50μL in a 96 well plate (Total volume 100 μL).
(5) It was left for 4 hours in a carbon dioxide incubator at 37 ° C.

2. Injury effect of macrophages co-cultured with Sato lung cancer cells on lung cancer cells
(1) In order to evaluate the damage effect on the cancer cells of macrophages activated by lipopolysaccharide and co-cultured with Sato lung cancer cells described in 1. from the amount of lactate dehydrogenase released from the cells into the culture solution, After centrifugation at 1,000 rpm for 5 minutes, 50 μL of the supernatant was dispensed into another 96-well plate.
(2). Subsequently, 50 μL of the substrate solution attached to the kit for measuring the amount of lactate dehydrogenase (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, cat. G1780) was added. In addition, as a positive control, a lactate dehydrogenase enzyme diluted solution (5000-fold diluted solution 50 μL) attached to the kit was used.
(3) Shaded with aluminum foil and left at room temperature for 30 minutes.
(4) 50 μL of stop solution was added to stop the reaction.
(5) The absorbance at 495 nm was measured using a microplate reader (Model 550, BIO-RAD).
(6). Cytotoxicity (%) was calculated from each absorbance.
(7) The cytotoxic effect of NR8383 on Sato lung cancer cells is shown in FIG. Compared with NR8383 not treated with lipopolysaccharide, NR8383 treated with lipopolysaccharide increased phagocytosis, and as a result, it was shown that the cytotoxic effect on Sato lung cancer was enhanced depending on the cell ratio.

Providing a therapeutic agent that enhances phagocytic activity of macrophages and induces cell death of macrophages that retain pathogens The therapeutic agent of this embodiment induces macrophages that retain pathogens to cell death by enhancing phagocytic activity of macrophages It is characterized by doing.

As shown in Table 2, lipopolysaccharide, which is well known as a macrophage activator, enhances macrophage phagocytic activity. In addition, interferon γ, which is a typical cytokine of macrophage activating factor, also enhances phagocytic activity. That is, enhancement of macrophage phagocytic activity is one of the indicators of macrophage activation. The enhancement of phagocytic activity by phagocytosis shown in this case can be said that stimulation of phagocytosis induced macrophage activation called enhancement of phagocytosis. In addition, it is known that the membrane structure of macrophages is changed with the activation of macrophages by lipopolysaccharide or interferon γ, and membrane-bound tumor necrosis factor (TNF) is induced in the macrophages. Therefore, membrane-bound TNF is induced with the activation of macrophages by stimulation of phagocytosis.

  AIDS develops when T cells are destroyed and the immune response is impaired by AIDS virus infection. By the way, AIDS virus infection infects not only T cells but also macrophages. This infection is due to the fact that the AIDS virus begins by adsorbing to the CD4 protein that is commonly expressed on the T cell and macrophage cell membranes. Macrophages infected with AIDS virus continuously produce AIDS virus without cell death II. As detailed in (2), it becomes an infectious agent medium.

  Therefore, if the macrophages infected with the AIDS virus can be induced to cell death, killing of the infectious agent medium will be achieved.

  As an example that can realize this possibility, it is shown that membrane-bound TNF can act on macrophage cells infected with AIDS virus (HIV) to specifically induce cell death.

Specific Application Example: Induction of HIV-infected cell death by membrane-bound TNF-expressing cells As an example in which TNF induces cell death in cells infected with pathogens, TNF against HIV-infected malt-4 (MOLT-4) cells Shows the effect.

1. Production of membrane-bound TNF-expressing cells
(1). In order to stably express membrane-bound TNF, mouse and human membrane-bound TNF expression plasmid (pMT2βG / Hu-proTNF).
(2) Then, this expression plasmid was mixed with a plasmid incorporating a neomycin resistance gene for selection of a transformant at a ratio of 10: 1 and introduced into mouse embryo-derived fibroblasts (NIH3T3 cells).
(3) The above-described transformed NIH3T3 cells were cultured for about 2 weeks in a carbon dioxide incubator at 37 ° C. in the presence of neomycin 800 μg / mL as a selection marker.
(4) After culture, a clone of TNF incorporated into the cell was obtained.
(5) It was confirmed by enzyme immunoassay that the obtained clones expressed membrane-bound TNF.

2. Preparation of HIV-infected malt 4 cells
(1). HIV-infected cells are malted in a medium (RPMI1640, 10% fetal bovine serum and penicillin (100 U / mL), streptomycin (100 μg / mL) added) in a carbon dioxide incubator at 37 ° C. 4 was infected with HIV-infected cells (HTLV-IIIB strain) according to the previous report (Journal of Biology 1889, 63, pp. 2504-2509). When an infection with AIDS virus is established in malt 4 cells, the malt 4 cells are characterized by continuously producing AIDS virus. Thus, when it comes to AIDS virus production, malt 4 cells are a model of AIDS virus-infected macrophages.
(2) Under this infection condition, 90% or more of the HIV-infected malt 4 cells became positive for the HIV antibody.
(3) Therefore, HIV is introduced into almost all malt-4 cells by HIV-infected cells, and HIV-infected malt-4 cells are prepared.

3. Cell death induction of HIV-infected cells by membrane-bound TNF-expressing cells
(1) NIH3T3 cells introduced with a TNF plasmid were cultured in a 24-well culture plate so that they were almost flush with each other.
(2) Next, a certain amount of HIV-infected malt 4 cells were added, and mixed culture was performed in a carbon dioxide incubator at 37 ° C. for 3 days.
(3) In order to examine the action of the expressed membrane-bound TNF on HIV-infected cells, the survival rate of HIV-infected cells was measured by the trypan blue dye exclusion method.
(4) As a result, 40% of malt 4 cells infected with HIV were found to die in mixed culture with membrane-bound TNF-expressing cells.

  As is clear from the above examples, it is clear that membrane-bound TNF induces cell death specifically in cells infected with AIDS virus (HIV). From this, it can be seen that the macrophages that became dysfunctional by retaining the pathogen are activated by phagocytosing the pathogen, and that the membrane-bound TNF induced as a result induces the pathogen-infected macrophages to cause cell death. it can.

Diseases caused by macrophages retaining pathogens include mycobacterial disease, AIDS, chlamydiasis, or toxoplasmosis. Mycobacterial diseases include mycobacterium tuberculosis or mycobacterium. Tuberculosis caused by Mycobacterium bovis, rye caused by MycobacteriumLeprae, atypical mycobacterial disease caused by Mycobacterium avium, etc. There are Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, etc. as the causative bacteria of chlamydiasis, any of which can effectively apply the present invention. By the way, the effect of killing M. tuberculosis in macrophages by the RFP-PLGA microparticles shown in this embodiment is that rifampicin is released from the RFP-PLGA microparticles taken into the phagosome by phagocytosis , permeates the phagosomal membrane, This is not achieved without the passage of at least two intracellular vesicular membrane structures that permeate the phagosomal membrane of Mycobacterium tuberculosis-containing vesicles. The survival strategies in the macrophages of the causative bacteria shown above are various. In general, mycobacteriosis, Legionella, and Toxoplasma survive in macrophages because the causative bacteria survive in the phagosome. Listeria and chlamydia have the characteristic that the causative bacteria escape from the phagosome. Intracellular survival of these causative bacteria is easier than the case where causative bacteria are present in the phagosome, since the drug can be expected to be effective once the drug penetrates the phagosomal membrane from the viewpoint of the arrival of the drug. It is estimated that. AIDS is similar to Listeria and the like in that a drug can be expected to reach the causative bacteria present in the cells. From the above, the present invention is a promising treatment for the causative bacteria described above.

Moreover, the chemical | medical agent with respect to each disease which can apply this invention effectively is listed below.
1) Mycobacteriosis: rifampicin, isoniazid, ethambutol, pyrazinamide, azithromycin, kanamycin, streptomycin sulfate, enbiomycin, ethionamide, cycloserine, levofloxacin, diaphenylsulfone.
2) AIDS: azidothymidine, dideoxyinosine.
3) Chlamydiasis: minocycline hydrochloride, doxycycline hydrochloride, clarithromycin, sparfloxacin, loxithromycin, levofloxacin.
4) Toxoplasmosis: Pyrimethamine, sulfamonomethoxine, acetylsulpyramycin.
5) Malaria: chloroquine phosphate, quinine sulfate, sulfadoxine, mefloquine.
6) Cancer: 5-fluorouracil, adriamycin, cisplatin, etoposide, mitomycin, vincristine, taxol, camptothecin, vinblastine, cyclophosphamide, bleomycin.
7) Crohn's disease: salazosulfapyridine, glucocorticoid.
8) Rheumatism: gold sodium thiomalate, penicillamine, bucillamine, glucocorticoid.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Industrial applicability

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a therapeutic agent that can effectively treat a disease affected by macrophage dysfunction or by mediating macrophages.

FIG. 1 is a diagram illustrating a comparison between the present invention and a conventional therapeutic drug in macrophages. FIG. 2 is a diagram showing a particle size distribution of the fine particle formulation according to the embodiment of the present invention. FIG. 3 is a diagram illustrating how the amount of rifampicin taken up by NR8383 cells differs according to the administration of the present invention and the administration of a conventionally used solution. FIG. 4 is a diagram (part 1) for explaining that the rifampicin retention ability of RFP-PLGA fine particles differs in the amount of rifampicin released depending on the composition of the PLGA fine particles. The experimental value includes an error of 5%. FIG. 5 is a diagram (part 2) for explaining that the rifampicin retention ability of RFP-PLGA fine particles differs in the amount of rifampicin released depending on the composition of the PLGA fine particles. The experimental value includes an error of 5%. FIG. 6 is a diagram illustrating that macrophages that have phagocytosed M. tuberculosis can kill M. tuberculosis in macrophages by phagocytosing RFP-PLGA microparticles. The survival rate was evaluated as 1: 5% or less, 2: 5 to 25%, 3:25 to 50%, 4:50 to 75%, 5: 75% or more. The administered rifampicin was 100 μg / mL for single administration (indicated by RFP in the figure) and 5 μg / mL (estimated value) for administration by RFP-PLGA (indicated by RFP-PLGA in the figure). FIG. 7 shows that the phagocytic ability of alveolar macrophage NR8383 is activated using lipopolysaccharide, thereby enhancing the cytotoxic effect of NR8383 in a dysfunctional state on Sato lung cancer cells by co-culture with Sato lung cancer cells. It is a figure explaining this. (□) shows the cytotoxic effect of NR8383 not treated with lipopolysaccharide on Sato lung cancer, (■) shows the cytotoxic effect of NR8383 treated with lipopolysaccharide (1 μg / mL) on Sato lung cancer cells (co-culture) 4 hours). The cytotoxic effect (%) was evaluated by calculating from the amount of lactate dehydrogenase released into the culture solution.

Claims (2)

Treatment for lung cancer characterized by containing lipopolysaccharide of Pantoea agglomerans, enhancing macrophage phagocytic activity, acting on dysfunctional macrophages, and having cytotoxic action on lung cancer cells medicine.   The therapeutic agent according to claim 1, wherein the macrophages are resident in mucosal tissues.

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