US20060105048A1 - Remedy - Google Patents

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US20060105048A1
US20060105048A1 US10/525,964 US52596405A US2006105048A1 US 20060105048 A1 US20060105048 A1 US 20060105048A1 US 52596405 A US52596405 A US 52596405A US 2006105048 A1 US2006105048 A1 US 2006105048A1
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macrophages
plga
cells
remedy
macrophage
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Hiroshi Terada
Kimiko Makino
Gen-Ichiro Soma
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Assigned to TERADA, HIROSHI reassignment TERADA, HIROSHI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SOMA, GEN-ICHIRO, MAKINO, KIMIKO
Publication of US20060105048A1 publication Critical patent/US20060105048A1/en
Priority to US12/659,796 priority Critical patent/US8425939B2/en
Priority to US13/831,439 priority patent/US20130202663A1/en
Priority to US14/221,606 priority patent/US20140206641A1/en
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61P37/02—Immunomodulators
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    • A—HUMAN NECESSITIES
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a remedy to normalize macrophages with dysfunction by taking advantage of the phagocytic capacity of macrophages, or effective for various infectious pathogens.
  • the remedy according to the invention is aimed at all substances given on a therapeutic and/or diagnostic basis and assemblies thereof, and a formulation thereof is a medical mixture of a medicament (including a medicament carrier in some cases) and the medicament carrier.
  • the macrophage is a cell which configures a mononuclear phagocyte system (MPS).
  • MPS mononuclear phagocyte system
  • a monocyte in blood is derived from a hematopoietic stem cell in bone marrow, divides/differentiates in the bone marrow to flow out into the blood, and settles in various tissues to differentiate a monocytic cell called by various names.
  • This cell is referred to as a histiocyte in connective tissue, Kupper cell in the liver, an alveolar macrophage in the lung, a macrophage in the lymph node and spleen, a thoracic macrophage/peritoneal macrophage in a body cavity, an osteoclast in bone, Langerhans cell in skin, a microglia cell in nerve tissue, a microglia in the brain, and an A type cell in synovial membrane, and has tissue-specific natures.
  • Phagocytosis is one of the best-known functions of the macrophage. This function may be believed to be one of the most basic functions which has been comprised since organisms were single-cell organisms in the process of evolution from the beginning of life. Therefore, one of features of the macrophage is that its existence has universality species-transversely. It seems that this feature provides one of the great advantages for research on macrophage functions. That is, if a remedy for diseases of mammals is developed/researched, macrophages from other than mammals can be functionally useful as research material. This is due to an aspect that the macrophage is a phylogenetically conserved cell.
  • a second important aspect as the macrophage functions is a host defense action.
  • This action is referred to as a non-specific host defense action, but recent research has demonstrated that the host defense action of the macrophage is also specific originally, the term, non-specificity has been a word corresponding to antigen specificity and immunological memory characterized for the T cell, and at present, it has not been proved that the macrophage has the antigen specificity or the immunological memory in an accurate sense. Thus, it is not exactly wrong that the host defense action of the macrophage is non-specific.
  • the response of macrophages is qualitatively different depending on types of pathogens, and a part of these qualitatively different responses correspond to a difference in receptors on a macrophage cell surface, which recognizes the pathogens.
  • the action of macrophages is specific.
  • the host defense action based on the macrophages As an innate immune system and the host defense action based on the T cells as an acquired immune system. Furthermore, considering the phylogenetic universality, it is a matter of course that the innate immune system is also a phylogenetically highly conserved host defense system.
  • the innate immune system based on the macrophages plays a central role in the host defense system which are the foreign substance discrimination and elimination system in not only species not having the acquired immune system but also species comprising the acquired immune system. Even in organisms comprising the acquired immune system, the function of the innate immune system covers the discrimination and elimination of foreign substances in nearly all cases, and in the case where this is insufficient, the acquired immune system is recruited. Even in this case, the antigen presentation by the macrophage is essential for the specific recognition of the foreign substance, and when eliminating the foreign substance, those which play central roles in the elimination system are the macrophages and the like which are the cells which configure the innate immune system.
  • cytotoxic T cells characteristic of the acquired immune system (e.g., elimination of cells infected with virus), but other T cells presented with the antigen by the macrophages are essential for proliferation and maturation of these cytotoxic T cells. That is, in order for the acquired immune system to significantly serve, the innate immune system must serve completely and suitably to the end.
  • Leukocyte adhesion deficiency Chediac-Higashi syndrome and the like are known as a phagocytic function abnormality as an abnormality of foreign substance phagocytosis.
  • the abnormality is observed in the phagocytic function, and in the latter, transport of a lysosomal enzyme to a phagocytic void cavity is abnormal, thus the disinfection capacity is reduced and the phagocytic capacity is remarkably facilitated.
  • Wiskott-Aldrich syndrome As the disease as the abnormality of the foreign substance elimination capacity and the immunological competence, Wiskott-Aldrich syndrome can be included.
  • the macrophages of the patient exhibit a complicated immunological abnormality such as migration abnormality and defect of an antibody dependent cytotoxic action.
  • MHC major histocompatibility complex
  • cytokines physiologically active substances which perform intercellular signaling play important roles in the host defense system based on the discrimination and elimination of a foreign substance.
  • the macrophages produce and secrete a wide variety of cytokines. In this way, the macrophage functions are essential for individual homeostasis also with respect to the discrimination and elimination of a foreign substance.
  • tissue-specific macrophages which are resident in various tissues and have inherent characters. This is obvious when observed in the mucosal tissue which is a point of contact between an individual and an environment. Specific macrophages are resident in submucous layers of the respiratory organ, digestive organ and genitourinary organ, respectively. These tissue-specific macrophages biologically respond to tissue-specific internal and external environments. This suggests that the macrophages play an important role for organism homeostasis in addition to the discrimination and elimination of a foreign substance. There are many unknown aspects in the physiological significance of the tissue-specific macrophages. On reflection, considering an existence significant of the tissue-specific macrophages from a new viewpoint, their relation to various pathologies maybe now brought to attention.
  • the dysfunction of the tissue-specific macrophage is involved in the induction of tissue-specific pathologies.
  • intractable diseases such as Crohn's disease which is one of the inflammatory intestinal diseases, further autoimmune disease such as rheumatoid, and aging diseases such as osteoporosis
  • the dysfunction of the macrophages is involved in some form.
  • acid-fast bacteria such as tuberculosis germs
  • problems of the acid-fast bacteria per se it may be thought that the dysfunction of the alveolar macrophages is present in the context of the pathology.
  • tuberculosis According to WHO, tuberculosis, AIDS, malaria and the like are chronic intractable diseases which must be considered to be the most important on a worldwide scale. For example, it is described that 800 million or more patients with tuberculosis occur annually and300 million die. It is an urgent task to develop a remedy (medicament/formulation) effective in response to these diseases, and its social significance is extremely great.
  • the macrophage With respect to infection defense of and elimination of a pathogen, one of the cells which plays the most important role in vivo is the macrophage.
  • the macrophages are distributed in all organs. These macrophages are different in morphology and functions depending on the organs where the macrophages exist, but they are common in the aspect that they perform the infection defense/elimination of the pathogen.
  • infectious pathogens have acquired various means for avoiding attack from the macrophages in the process of evolution. Furthermore, the infectious pathogens are often hidden in the macrophages to make the macrophage a host. It is a matter of course that the pathogen which has succeeded to parasitize in the macrophage in this way causes an infectious disease chronically and repeatedly, and it is not unusual to result in a fatal consequence. That is, in this case, the macrophage which should primarily accomplish the infection defense/elimination of a pathogen serves as an infectious pathogen vehicle in reverse.
  • the pathogen Mycobacterium tuberculosis or Mycobacterium bovis
  • Mycobacterium tuberculosis or Mycobacterium bovis is phagocytosed by the macrophage in the pulmonary alveolus which is an infectious pathway in an early stage, and is stably present in a phagosome formed at that time. That is, the pathogen can live by making the macrophage which should primarily digest it as a “shelter”.
  • causative pathogens such as Mycobacterium leprae which is a causative pathogen of lepra, Mycobacterium avium which is a causative pathogen of atypical mycobacteriosis, Chlamydia pneumoniae, Chlamydia trachomatis or Chlamydia psittaci which is a causative pathogen of chlamydiosis of the intractable diseases whose radical therapy has not been established and propagation has been feared are common in the aspect that the macrophage becomes the infectious pathogen vehicle.
  • the macrophages as the pathogenvehicles are still alive as pathogen sources and continue to supply the pathogens.
  • the above is a reason why currently there is no radical therapy for the pathogen which parasitizes in the macrophage.
  • the invention makes this point a subject.
  • it is possible to exterminate the macrophages infected with the pathogens as the pathogen vehicles or exterminate the pathogens in the macrophages infected with the pathogens many chronic intractable infectious diseases described above can be radically treated. Therefore, it is an object of the invention to provide a remedy for a disease caused based on dysfunction of the macrophage or by making a macrophage a vehicle.
  • the present inventors have led to a remarkably novel idea where it is an object to exterminate macrophages infected with pathogens as pathogen vehicles, exterminate the pathogens in the macrophages infected with the pathogens and act upon macrophages whose function has become abnormal due to a disease, and have completed the invention.
  • a remedy of the invention is characterized by facilitating a phagocytic activity of macrophages and exterminating pathogens in the macrophages.
  • the remedy of the invention is characterized by facilitating the phagocytic activity of macrophages and leading the macrophages to cell death.
  • the remedy of the invention is characterized by facilitating the phagocytic activity of macrophages and acting upon macrophages in a dysfunctional state.
  • the remedy of the invention is for any of mycobacteriosis, AIDS, chlamydiosis or toxoplasmosis. This enables to effectively treat the diseases where the macrophages retain the pathogens.
  • the remedy of the invention is for Crohn's disease, rheumatoid, cancer or immunodeficiency syndrome. This enables to effectively treat the diseases where the macrophages are in a dysfunctional state. AIDS is included in the immunodeficiency syndrome.
  • the above macrophages are those which are resident in mucosal tissues. This enables to effectively treat the disease at sites such as the respiratory organ, digestive organ and genitourinary organ where the pathogens cause primary infection.
  • the above macrophages are those which are resident in any of the peritoneal cavity, greater omentum, milky spot, pulmonary alveolus, pulmonary stroma, liver, portal vein area, spleen, bone marrow, thymus, digestive tract, palatine tonsil, adrenal gland, pituitary, thyroid stroma, Langerhans islet, parathyroid gland, pineal gland, testis, ovary, oviduct, uterus, placenta, skin, meningis, brain substance and choroid plexus, or that the above macrophages are microglia, precursor cells of microglia, glia cells, precursor cells of glia cells, precursor cells of the above resident macrophages, analogous cells of the above resident macrophages, orprecurs or cells of the above resident macrophage analogous cells. This enables to effectively treat the diseases caused in various organs and tissues in the body.
  • the remedy of the invention is characterized by containing PLGA [poly(lactic acid/glycolic acid) copolymer] and being in response to the tuberculosis.
  • PLGA with a molecular weight of 1,500 to 150,000. This enables to provide a fine particle formulation which is biodegradable and is phagocytosed by the macrophages.
  • PLGA with a molecular weight of 1,500 to 75,000. This enables to provide a fine particle formulation which is phagocytosed by the macrophages and easily releases a medicament in the macrophages.
  • PVA polyvinyl alcohol
  • PEG polyethyleneglycol
  • PEO polyethylene oxide
  • sugar protein, peptide, phospholipid, or cholesterol.
  • PVA poly(ethylene glycol)
  • PEG poly(ethylene glycol)
  • PEO poly(ethylene glycol)
  • sugar protein
  • peptide phospholipid
  • cholesterol a fine particle formulation where major particle diameters are 1 to 6 ⁇ m. This enables to effectively take advantage of a phagocytic function of the macrophage to incorporate in the macrophages.
  • FIG. 1 is a view illustrating by comparison a drug concentration in a macrophage of a remedy of the invention with that of a conventional remedy.
  • FIG. 2 is a view showing a particle diameter distribution of a fine particle formulation in an embodiment mode of the invention.
  • FIG. 3 is a view illustrating by comparison how much amounts incorporated in NR8383 cells are different by administering rifampicin according to the invention and by administering it using a solution conventionally used.
  • FIG. 4 is a view (No. 1) illustrating a rifampicin-retaining capacity of RFP-PLGA fine particles, i.e., that amounts of rifampicin released are different depending on compositions of PLGA fine particles. Experimental values contain an uncertainty of 5%.
  • FIG. 5 is a view (No. 2) illustrating a rifampicin-retaining capacity of RFP-PLGA fine particles, i.e., that amounts of rifampicin released are different depending on compositions of PLGA fine particles. Experimental values contain an uncertainty of 5%.
  • FIG. 6 is a view illustrating that tuberculosis germs in the macrophages can be exterminated by phagocytosis of RFP-PLGA particles by the macrophages which have phagocytosed the tuberculosis germs. Viability was evaluated by ranking as follows: 1: 5% or less, 2: 5 to 25%, 3: 25 to 50%, 4: 50 to 75%, and 5: 75% or more.
  • An amount of administered rifampicin is 100 ⁇ g/mL in a single administration (indicated by RFP in the figure) or 5 ⁇ g/mL (estimated value) in the administration by RFP-PLGA (indicated by RFP-PLGA in the figure).
  • FIG. 7 is a view illustrating that a cytotoxic effect of alveolar macrophages NR8383 in a dysfunctional state by co-culture with Sato lung cancer cells on the Sato lung cancer cells is enhanced by activating a phagocytic capacity of NR8383 using lipopolysaccharide.
  • Open squares indicate the cytotoxic effect of NR8383 without treatment with lipopolysaccharide on the Sato lung cancer cells
  • solid squares indicate the cytotoxic effect of NR8383 treated with lipopolysaccharide (1 ⁇ g/mL) on the Sato lung cancer cells (co-cultured for 4 hours).
  • the cytotoxic effect (%) was evaluated by calculating amounts of lactate dehydrogenase released in media.
  • the phagocytosis is a function in which a solid with a size of about 1 ⁇ m or more is actively incorporated in a macrophage cell.
  • a solid made artificially is actively phagocytosed, it is possible to accumulate the solid in the macrophage at a concentration which cannot be usually accomplished.
  • Such a solid can be generally provided as a particle, but to actively phagocytose, it is necessary to optimize a particle diameter, particle surface property (having a charge, having a certain flexible structure, etc,) advantageous for phagocytosis.
  • the medicament is also actively incorporated in the macrophage.
  • An essential part of the invention is in the aspect that diseases due to dysfunction of the macrophage (I. 2. (5), (7), II. (1), mycobacteriosis, AIDS, chlamydiosis, toxoplasmosis, cancer and the like) are treated by taking advantage of the phagocytic function that the macrophages have.
  • diseases due to dysfunction of the macrophage I. 2. (5), (7), II. (1), mycobacteriosis, AIDS, chlamydiosis, toxoplasmosis, cancer and the like
  • the above object is accomplished by preparing a formulation in which a medicament effective for them is contained in fine particles (medicament carrier) which can be phagocytosed by the macrophages.
  • the formulation which is a medical mixture of the medicament carrier and the medicament
  • the invention has a novelty in the aspect of actively taking advantage of phagocytic activity of the macrophage based on an idea which completely reverses the conventional beliefs.
  • phagocytosis As one of the host defense functions of macrophages, there is the phagocytosis.
  • the phagocytosis is an inherent function characteristically observed in the macrophages, and it is possible to incorporate particles with a size which cannot be incorporated by cells other than the macrophages.
  • Pathological microorganisms such as bacteria are phagocytosed by the macrophages and decomposed in the macrophages. Therefore, one of biological significance of the phagocytic function of the macrophages is to impair the pathological microorganisms.
  • the macrophages are activated by the phagocytosis and become able to oppose pathological microorganisms in some cases.
  • a particle diameter is 1 to 6 ⁇ m. Such a particle is referred to as a fine particle.
  • a particle surface is wetted with a medium (body fluid around the macrophages in vivo) of macrophages, but the particle is not immediately dissolved and is present as the particle for a certain time period.
  • the particle is a solid in a temperature range of 20 to 45° C.
  • a specific gravity of the particle is larger than that of the medium (body fluid around the macrophages in vivo) of macrophages.
  • the particle has a surface layer through which water and ions are permeable.
  • the particle surface is required to have a macromolecular layer with high histocompatibility. It is necessary to retain a particle form until being incorporated in the macrophage while being metabolized by decomposing into components non-toxic for the body in vivo (degradability in vivo) after being incorporated in the macrophage or when not being incorporated.
  • poly(lactic acid/glycolic acid)copolymer hereinafter referred to as “PLGA”) or polylactic acid (hereinafter referred to as “PL”) is a candidate.
  • PL is more hydrophobic and requires a longer time until being decomposed than PLGA.
  • PLGA changes the decomposing rate depending on the monomer ratio. The larger the molecular weight is, the longer time it takes until being decomposed.
  • the molecular weight of PLGA is 1,000 or less, there is a possibility that it is present as a liquid in the temperature range of 20 to 45° C. Therefore, the molecular weight of PLGA which is present as a solid in the temperature range of 20 to 45° C. is desirably 1,500 or more.
  • a medicament contained in the PLGA particle from the particle in the case of PLGA with a molecular weight of about 20,000, the medicament is released in an almost zero order. That is, a released amount is always retained constantly.
  • a pulse type release where the medicament is released after a constant time period rather than the zero order release has been observed.
  • the time period where the pulse type of a medicament release is observed is delayed in the PLGA formulation with a large molecular weight. That is, a pattern of the medicament release from the PLGA particles depends on the molecular weight of PLGA.
  • the decomposing rate of PLGA related to the medicament release is not only delayed along with molecular weight increase of PLGA, but also the rate is faster in vivo such as in the macrophage than in vitro, and thus it appears that it is possible to effectively use those having a range of molecular weight up to 150,000.
  • a fine particle formulation with particle diameters of 1 to 6 ⁇ m made up of PLGA where a molecular weight is 1,500 to 150,000 and a monomer ratio of lactic acid/glycolic acid is 50:50 to 75:25 (acceptable range) and a fine particle formulation with particle diameters of 1 to 6 ⁇ m made up of PLGA where a molecular weight is 5,000 to 75,000 and a monomer ratio of lactic acid/glycolic acid is 50:50 to 75:25 (suitable range) are easily phagocytosed by the macrophages and are optimal for the object that the medicament is released while holding a constant persistence from the fine particle formulation which internally includes the medicament in the macrophages.
  • the particles In order for the rifampicin-containing PLGA particles to elicit the antituberculous action, the particles must have appropriate degradability and a high rate of internally included rifampicin.
  • the degradability and the internally included rate are reduced in the PLGA particles with large molecular weight, and thus, it appears that because of the use of PLGA with high molecular weight of 82,500, the antituberculous action was not elicited.
  • the particles have not been actively incorporated by the macrophages, or no formulation where the activation is intended has been made, and in fact, no phagocytic rate of the rifampicin-containing PLGA particles has been examined.
  • the concentration of rifampicin in the macrophages in vitro using the rifampicin-containing PLGA particles described in this report is only 0.45 ⁇ g/10 6 cells.
  • the concentration of rifampicin in the macrophages reaches 6 ⁇ g/10 6 cells which is 13 times or more.
  • the macrophages are highly tissue-specific, and thus it is a well-known fact that the result obtained using the macrophages present in blood cannot apply to the tissue-specific macrophages, e.g., the alveolar macrophages. That is, as a novelty of the invention, the novelty of the concept is a matter of course.
  • Examples which support this concept include the aspects that the alveolar macrophages are used and the phagocytic activity of the cells is induced, and the high medicament concentration is actually retained in the alveolar macrophages, and the rifampicin-containing PLGA particles are produced and provided where the extermination of tuberculosis germs in the alveolar macrophages by the formulation is obviously more excellent than that by rifampicin alone.
  • a formulation in which a medicament of type (2) or (3) is contained in a remedy of type (1) is effective. That is, in order for the remedy of type (2) or (3) to effectively act in the macrophages, the remedy of type (1) facilitates the phagocytic activity of the macrophages to enhance the function which carries the remedy of type (2) or (3) into the macrophages. That is, as shown in FIG. 1 , the remedy intended by the invention is easily phagocytosed by the macrophages, facilitates the phagocytic activity by being phagocytosed, and thus the concentration of the remedy in the macrophages becomes remarkably higher compared to the case of administering the remedy alone.
  • Tuberculosis is set forth as one example with respect to the effectiveness of the remedy presented in the invention.
  • Tuberculosis germs invade from the respiratory tract to pulmonary alveoli by droplet, and are phagocytosed by alveolar macrophages. Typically, phagocytosed pathogens are destined to be decomposed by an attack of protease in cells. However, the tuberculosis germs avoid the attack of protease and are alive in the macrophages. These tuberculosis germs in the macrophages migrate out of the macrophages, and persistently supply the tuberculosis germs in a host body.
  • medicaments such as isoniazid, rifampicin, streptomycin sulfate and ethambutol are used. All medicaments are effective for the tuberculosis germs out of the macrophages, but do not exhibit any effect on the tuberculosis germs in the alveolar macrophages. This is mainly attributed to that a medicament concentration sufficient to exterminate the tuberculosis germs is not obtained in the alveolar macrophages.
  • the medicament concentration sufficient to exterminate the tuberculosis germs is obtained in the alveolar macrophages by taking advantage of the phagocytosis of the alveolar macrophages, it is also possible to exterminate the tuberculosis germs in the alveolar macrophages.
  • the phagocytosis is utilized in order to selectively increase the medicament concentration in the macrophages.
  • PLGA a method for producing a remedy which enhances the phagocytic capacity of macrophages and effects thereof are described.
  • PLGA poly(lactic acid/glycolic acid)copolymer
  • monomer ratio 75:25
  • molecular weight 20,000 (Wako Pure Chemical Industries Ltd., PLGA-7520)
  • a fine particle formulation is precipitated by centrifugation (3,000 rpm, 15 minutes), separated, and further washed twice by adding 10 mL of distilled water using a centrifuge.
  • a particle diameter distribution of the resulting fine particle formulation is shown in FIG. 2 .
  • the prepared fine particle formulation has a peak at a diameter of about 2 ⁇ m and has a distribution between 1 to 10 ⁇ m.
  • This particle is a solid at ambient temperatures.
  • a yield calculated from PLGA (500 mg) used for the preparation and an entire weight of the recovered formulation was about 90%.
  • PLGA-5005 (PLGA, molecular weight: 5,000; lactic acid/glycolic acid: 50:50)
  • PLGA-5010 (PLGA, molecular weight: 10,000; lactic acid/glycolic acid: 50:50)
  • PLGA-5020 (PLGA, molecular weight: 20,000; lactic acid/glycolic acid: 50:50)
  • PLGA-7505 (PLGA, molecular weight: 5,000; lactic acid/glycolic acid: 75:25)
  • PLGA-7510 (PLGA, molecular weight: 10,000; lactic acid/glycolic acid: 75:25)
  • the method for preparing the PLGA fine particle formulation is the same as 1. (b)(1) to (6) except the kind of PLGA.
  • An average particle diameter of the resulting fine particle formulation was about 2 ⁇ m even when any PLGA was used.
  • a yield calculated from PLGA (500 mg) used for the preparation and an entire weight of the recovered formulation was about 90%.
  • Alveolar macrophage cells (NR8383 cells ) are prepared at 1 ⁇ 10 6 cells/mL in a medium (Ham F-12K, 15% fetal calf serum), added in a 24-well plate, and 0.04, 0.4 or 4 ⁇ g of PLGA-7520 fine particle formulation is added thereto, which is then cultured in a carbon dioxide gas incubator (37° C.).
  • PBS trypsin/phosphate buffer
  • FITC-PSLP Polystyrene latex particles with particle diameter of 2.0 ⁇ m labeled with fluorescein isothiocyanate (FITC) at 1 ⁇ 10 7 are added, and a mixture is cultured for one hour. Labeling with fluorescent FITC is for easily performing quantitative analysis.
  • a change of FITC-PSLP phagocytosed amounts in the macrophages by the PLGA fine particle formulation is as shown in Table 1.
  • a low amount addition of the PLGA fine particle formulation does not remarkably affect the phagocytic capacity of the macrophages, but it is evident that the addition of 0.4 ( ⁇ g/mL) noticeably activates the phagocytic capacity.
  • TABLE 1 Enhancement effect of PLGA fine particle formulation on phagocytic capacity of macrophages Amount of PLGA fine particle Uptake rate of formulation ( ⁇ g/mL) FITC-PSLP (%) 0 11.6 0.004 8.8 0.04 12.3 0.4 20.2 II. Enhancement of Phagocytic Capacity of Macrophages by Lipopolysaccharide 1.
  • Pantoea agglomerans belonging to gram negative bacterium, genus Pantoea
  • Pantoea agglomerans is added to 7 liters of bouillon (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl, 1 g/L of glucose, pH 7.5), cultured with shaking at 35° C. for 24 hours, and about 70 g of wet bacterial body is collected.
  • a resulting crude frozen/dried lipopolysaccharide is dissolved in distilled water, applied on an anion exchange chromatography (supplied from Pharmacia, Q-Sepharose Fast Flow), a sample solution is passed through a column using a buffer containing 10 mM of Tris-HCl (pH 7.5) and 10 mM of NaCl, and a Limulus activity fraction is eluted with 200 to 400 mM of NaCl/10 mM Tris-HCl (pH7.5). By ultrafiltrating this eluted solution, desalting, concentrating and freezing/drying under the same condition as the above, it is possible to yield about 300 mg of purified lipopolysaccharide from about 70 g of the wet bacterial body.
  • Alveolar macrophage cells (NR8383) are prepared at 1 ⁇ 10 6 cells/mL in a medium (Ham F-12K, 15% fetal calf serum) and added to a 24-well plate. Lipopolysaccharide is added thereto to become 1 ⁇ g/mL, and the culture is performed in a carbon dioxide gas incubator (37° C.).
  • the cells present on the liquid surface are collected, and washed twice with PBS. Subsequently, 1 mL of the medium is added, FITC-PSLP with a particle diameter of 2.0 ⁇ m at 1 ⁇ 10 7 is added, which is then cultured for one hour.
  • PLGA poly(lactic acid/glycolic acid)copolymer
  • PLGA 500 mg
  • Rifampicin (0, 50, 100 or 200 mg) are dissolved in 1.5 mL of methylene chloride.
  • a fine particle formulation is precipitated by centrifugation (3,000 rpm, 15 minutes), separated, and further washed twice by adding 10 mL of distilled water using a centrifuge.
  • RFP-PLGA 5005 (PLGA, molecular weight: 5,000; lactic acid/glycolic acid: 50:50).
  • RFP-PLGA 5010 (PLGA, molecular weight: 10,000; lactic acid/glycolic acid: 50:50).
  • RFP-PLGA 5020 (PLGA, molecular weight: 20,000; lactic acid/glycolic acid: 50:50).
  • RFP-PLGA 7505 (PLGA, molecular weight: 5,000; lactic acid/glycolic acid: 75:25).
  • RFP-PLGA 7510 (PLGA, molecular weight: 10,000; lactic acid/glycolic acid: 75:25).
  • RFP-PLGA 7520 (PLGA, molecular weight: 20,000; lactic acid/glycolic acid: 75:25).
  • the RFP-PLGA 7510 fine particle formation was prepared.
  • the membrane emulsification method is an emulsification method where one (dispersion phase) of two kinds of liquids which do not blend together is pressed to disperse in another liquid (continuous phase) through a porous glass membrane. By the use of this method, it is possible to obtain an emulsion with uniform particle diameters.
  • PLGA poly(lactic acid/glycolic acid)copolymer
  • a fine particle formulation is precipitated by centrifugation (3,000 rpm, 15 minutes), separated, and further washed twice by adding 10 mL of distilled water using a centrifuge.
  • An average particle diameter of the resulting fine particle formulation is 1.98 ⁇ m.
  • a yield of PLGA calculated from PLGA (500 mg) used for the preparation and an entire weight of the recovered formulation was about 90%.
  • the yield of rifampicin was about 75%. This particle is a solid at ambient temperatures.
  • RFP-PLGA fine particles (molecular weight and composition) other than RFP-PLGA7510 fine particle formulation were collectively shown below.
  • RFP-PLGA 5005 (PLGA molecular weight: 5,000; lactic acid/glycolic acid 50:50)
  • RFP-PLGA 5010 (PLGA molecular weight: 10,000; lactic acid/glycolic acid 50:50)
  • RFP-PLGA 5020 (PLGA molecular weight: 20,000; lactic acid/glycolic acid 50:50)
  • RFP-PLGA 7505 (PLGA molecular weight: 5,000; lactic acid/glycolic acid 75:25)
  • RFP-PLGA 7520 (PLGA molecular weight: 20,000; lactic acid/glycolic acid 75:25)
  • the method for preparing these RFP-PLGA fine particle formulations is the same as the above membrane emulsification method except kinds of PLGA.
  • the average particle diameters of the resulting fine particle formulations are 2.20 ⁇ m in PLGA 5005, 2.66 ⁇ m in PLGA 5010, 2.29 ⁇ m in PLGA 5020, 2.00 ⁇ m in PLGA 7505, and 1.85 ⁇ m in PLGA 7520. All yields of PLGA calculated from PLGA (500 mg) used for the preparation and an entire weight of the recovered formulation were about 90%. The yields of rifampicinwere about 87% in PLGA 5005, about 78% in PLGA 5010, about 67% in PLGA 5020, about 91% in PLGA 7505, and about 58% in PLGA 7520.
  • Respective RFP-PLGA fine particles (50 mg) prepared by the membrane emulsification method were dispersed in 5 mL of phosphate buffer at pH 7.4 (ionic strength: 0.154 M) retained at 37° C. (temperature).
  • Rates of rifampicin released from respective RFP-PLGA fine particles into the supernatant are shown in FIG. 4 and FIG. 5 . From data of the present experiment, it was shown that releasing rates of rifampicin from PLGA with molecular weights of 5,000 and 10,000, i.e., PLGA 5005, PLGA 5010, PLGA 7505 and PLGA 7510 were fast. From these results, it has been shown that the PLGA compositions in which the molecular weight is 5,000 to 10,000 and the ratio of lactic acid to glycolic acid is 50:50 or 75:25 are excellent in migration of the drug into the macrophages via the phagocytosis.
  • the prepared RFP-PLGA fine particle formulation is dispersed again in PBS, the centrifugation (400 rpm, 5 minutes) is performed to eliminate large particles, and the fine particle formulation (about 30% in weight) prepared in sizes of about 1 to 3 ⁇ m by microscope observation are used for the following experiments.
  • DMSO dimethylsulfoxide
  • the cells are cultured for 12 hours, subsequently the cells (5 ⁇ 10 5 cells) are collected, and rifampicin in the cells is extracted with methylene chloride.
  • PLGA poly(lactic acid/glycolic acid)copolymer
  • PLGA 500 mg and rifampicin 100 mg are dissolved in 1.5 mL of methylene chloride.
  • a fine particle formulation is precipitated by centrifugation (3,000 rpm, 15 minutes), separated, and further washed twice by adding 10 mL of distilled water using a centrifuge.
  • the fine particle formulation is dispersed again in PBS, the centrifugation (400 rpm, 5 minutes) is performed to eliminate large fine particle formulation, and the fine particle formulation prepared in sizes of about 1 to 3 ⁇ m is used for the following experiments.
  • Alveolar macrophage cells (NR8383) are prepared at 1 ⁇ 10 6 cells/mL in a medium (Ham F-12K, 15% fetal calf serum), added in a 24-well plate, and 0.012, 0.12 or 1.2 ⁇ g of PLGA fine particle formulation is added thereto, which is then cultured in a carbon dioxide gas incubator (37° C.).
  • a medium Ham F-12K, 15% fetal calf serum
  • the cells are transferred to 1.5 mL of a sample tube, the medium is removed, 0.1 mL of 0.25% trypsin/PBS is added, amixture is left at roomtemperature for 5 minutes, then the culture supernatant is removed, and washing is performed.
  • 0.1 mL of 10% formalin/PBS is added to the cells, is fixed for 5 minutes, then 1 mL of distilled water is added, and then the centrifugation (700 rpm, 5 minutes) is performed, 1 mL of distilled water is added again, and the supernatant is removed. Thereto, 0.1 mL of distilled water is added to suspend the cells, 0.02 mL of the suspension is taken, spread on a slide glass, and dried.
  • the rifampicin-containing PLGA fine particle formulation activates the phagocytic capacity of the macrophages resulting in the increased rifampicin concentration in the macrophages, and thus makes it possible to accomplish the object of the invention to efficiently affect against the pathogens retained in the macrophages.
  • the above results indicate one example of the novel remedy where the remedy which acts upon the pathogens in the macrophages is effectively incorporated in the macrophages by “facilitating the phagocytic activity of the macrophages” which is a basic concept of the invention.
  • NR8383 cells at a concentration of 1 ⁇ 10 6 cells/mL were placed in a 6-well plate (total volume 5 mL).
  • the tuberculosis germs (BCG) at 10 per cell (multiplicity of infection (MOI) 10) were added to a cell culture medium.
  • NR8383 cells at a concentration of 1 ⁇ 10 6 cells/mL were seeded in a 24-well plate.
  • Respective RFP-PLGA fine particles at 10 per cell were added to the culture medium of NR8383 cells.
  • Rates of living bacteria and dead bacteria were measured using a staining method for fluorescein diacetate (FDA)/ethidium bromide (EB).
  • FDA fluorescein diacetate
  • EB was dissolved in PBS at a concentration of 20 ⁇ g/mL, and 50 ⁇ l thereof was diluted with 1 mL of PBS at use.
  • Equal amounts of diluted FDA and EB were mixed and used for staining. This mixed solution (1 ⁇ L) was placed on a slide glass, 1 ⁇ L of the bacterial solution was placed thereon, which was then left at room temperature for 2 minutes and observed under a fluorescent microscope.
  • FDA is decomposed by esterase activity derived from the bacteria to emit fluorescence with a green color.
  • esterase activity derived from the bacteria to emit fluorescence with a green color.
  • staining with FDA is not observed, and the bacteria are stained with EB to emit fluorescence with an orange color.
  • the RFP-PLGA fine particles were administered to the macrophages (macrophages infected with tuberculosis germs) which have previously phagocytosed the tuberculosis germs, and these fine particles were phagocytosed by the macrophages.
  • the administration of the RFP-PLGA fine particles to the alveolar macrophages infected with tuberculosis germs exterminated the tuberculosis germs in the cells at an efficiency of 20 times or more compared to the administration of rifampicin alone. That is, by administrating the drug via the phagocytosis of the particles, a treatment coefficient was improved by 20 times or more regardless of the antituberculous effect of the drug per se.
  • the phagocytosis by the alveolar macrophages was enhanced by 166% by the treatment with lipopolysaccharide (1 ⁇ g/mL), and the alveolar macrophages were activated by the lipopolysaccharide.
  • the alveolar macrophages whose phagocytic capacity has been activated by the lipopolysaccharide elicit the cytotoxic effect on lung cancer cells. Therefore, here, an example of the cytotoxic effect on lung cancer cells of the alveolar macrophages activated by the lipopolysaccharide is shown.
  • the cytotoxic effect on lung cancer cells by activating the alveolar macrophages is shown as the application example.
  • NR8383 cells at a concentration of 1 ⁇ 10 6 cells/mL were placed in a 24-well plate (total volume: 1.5 mL).
  • total volume 1.5 mL.
  • 5% fetal calf serum, 150 ⁇ l of F-12K medium, and 150 ⁇ l of 10 ⁇ g/mL lipopolysaccharide were added.
  • Sato lung cancer cells were prepared at a concentration of 1 ⁇ 10 5 cells/mL, and 50 ⁇ l per well was added in a 96-well plate.
  • NR8383 cells treated in the section (1) were prepared at concentrations of 5 ⁇ 10 5 , 1 ⁇ 10 5 , 5 ⁇ 10 4 and 1 ⁇ 10 4 cells/mL, and 50 ⁇ l per well was added to the 96-well plate (total volume: 100 ⁇ l).
  • FIG. 7 The cytotoxic effect of NR8383 on Sato lung cancer cells is shown in FIG. 7 . It has been shown that the phagocytosis by NR8383 treated with lipopolysaccharide is facilitated compared to NR8383 without treatment with lipopolysaccharide and consequently the cytotoxic effect on Sato lung cancer is enhanced depending on cell ratios.
  • the remedy in the present embodiment mode is characterized in that the macrophages which retain the pathogens are led to cell death by facilitating the phagocytic activity of the macrophages.
  • the lipopolysaccharide well-known as a macrophage activator facilitates the phagocytic activity of the macrophages.
  • interferon- ⁇ which is a representative cytokine of macrophage activating factors facilitates the phagocytic activity. That is, the facilitation of phagocytic activity of the macrophages is one of the indicators of the macrophage activation. It can be said that the facilitation of the phagocytic activity by the phagocytosis is the facilitation of macrophage activation which is the facilitation of phagocytosis by stimulation which is the phagocytosis.
  • TNF tumor necrosis factor
  • AIDS occurs due to destruction of T cells by infection with the AIDS virus resulting in immunodeficiency.
  • AIDS virus infects not only T cells but also macrophages. This infection initiates by adhesion of the virus to CD4 protein commonly expressed on T and macrophage cell membranes.
  • the macrophage infected with the AIDS virus does not lead to the cell death, but produces the AIDS virus persistently, and becomes the infectious pathogen vehicle as described in detail in II. (2).
  • the membrane-bound TNF can act upon the macrophage cells infected with the AIDS virus (HIV) to specifically lead the cells to cell death.
  • TNF induces cell death in the cells infected with pathogens
  • MOLT-4 cells infected with HIV the effect of TNF on MOLT-4 cells infected with HIV is shown.
  • this plasmid was mixed with a plasmid for selection of transformants, in which a neomycin resistant gene is incorporated at a ratio of 10:1, and introduced into fibroblast cells (NIH3T3 cells) derived from murine embryo.
  • NIH3T3 cells transformed were cultured in the presence of 800 ⁇ g/mL of neomycin which is a selection marker in the carbon dioxide gas incubator at 37° C. for about 2 weeks.
  • MOLT-4 cells were infected in the medium (RPMI1640, adding 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 ⁇ g/mL)) in the carbon dioxide gas incubator at 37° C. according to the previous report (Journal of Virology, Vol. 63, pp. 2504-2509, 1989) using cells infected with HIV (HTLV-IIIB strain).
  • MOLT-4 cells exhibit characteristics to persistently produce the AIDS virus. Therefore, with respect to the production of the AIDS virus, a MOLT-4 cell is a model of the macrophage infected with AIDS virus.
  • NIH3T3 cells where the plasmid of TNF had been introduced were semi-confluently cultured in a 24-well culture plate.
  • MOLT-4 cells infected with HIV was added and mixed/cultured in the carbon dioxide gas incubator at 37° C. for 3 days.
  • the membrane bound TNF induces specifically cell death in the cells infected with AIDS virus (HIV). From these phenomena, it can be seen that the macrophages led to dysfunction by retaining the pathogens are activated by phagocytosing the pathogens and consequently induced membrane bound TNF leads the macrophages infected with pathogens to cell death.
  • HIV AIDS virus
  • Mycobacteriosis includes tuberculosis whose causative pathogen is Mycobacterium tuberculosis or Mycobacterium bovis, lepra whose causative pathogen is Mycobacterium leprae, or atypical mycobacteriosis whose causative pathogen is Mycobacterium avium and the like, or the like.
  • Chlamydia pneumoniae As causative pathogens of chlamydiosis, there are Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci and the like.
  • the present invention can effectively apply to all of them.
  • the exterminating effect of the RFP-PLGA fine particles on the tuberculosis germs in the macrophages shown in the present embodiment mode is not accomplished unless rifampicin is released from the RFP-PLGA fine particles incorporated in phagosomes by phagocytosis and at least two passages via intracellular vesicular membrane structures are possible where the rifampicin permeates the phagosome membrane and further permeates the phagosome membrane of the phagosome which contains the tuberculosis germs. Survival strategies of the causative pathogens shown above in the macrophages are diverse.
  • the causative pathogens are alive in the macrophages by surviving in the phagosomes.
  • Listeria and Chlamydia have the characteristics that the causative pathogens evacuate from the phagosomes.
  • This intracellular existence aspect of the causative pathogens is presumed to be more easily compared to a case where the causative pathogens exist in the phagosomes because the drug efficacy can be anticipated when the drug permeates the phagosome membrane once from the perspective of delivery of the drug.
  • AIDS is similar to Listeria and the like in the aspect that drug efficacy can be anticipated if the drug is delivered to the causative pathogens present in the cells. From the above, the invention is a promising treatment for the causative pathogens described above.
  • Mycobacteriosis rifampicin, isoniazid, ethambutol, pyrazinamide, azithromycin, kanamycin, streptomycin sulfate, enviomycin, ethioniamide, cycloserine, levofloxacin, diaphenylsulfone.
  • Chlamydiosis minocycline hydrochloride, doxycycline hydrochloride, clarithromycin, sparfloxacin, roxithromycin, levofloxacin.
  • Toxoplasmosis pyrimethamine, sulfamonomethoxine, acetylspiramycin.
  • Rheumatoid gold sodium thiomalate, penicillamine, bucillamine, glucocorticoid.

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