JP4834829B2 - Tryptophan metabolic intermediates effective in suppressing Chlamydia spp. - Google Patents

Description

The present invention relates to an oral or injectable pharmaceutical composition for suppressing infection or growth of Chlamydia bacteria. More specifically, the present invention relates to a pharmaceutical composition comprising 5- hydroxytryptophan , melatonin, serotonin and pharmacologically acceptable salts, esters or derivatives thereof as components .

  Chlamydiaceae is an obligate intracellular parasitic bacterium that can grow only in cells of eukaryotes such as humans. Chlamydia trachomatis (Chlamydia trachomatis) is mainly a disease. Infecting the genital organs and becoming the first cause of sexually transmitted diseases in Japan, it has become widely known. Chlamydia pneumoniae (Chlamydophila pneumoniae) mainly infects inflammatory sites in the lungs and arteries and is a causative agent of colds, and at the same time, has been pointed out to be associated with arteriosclerosis and ischemic heart disease. Chlamydia pitaci is known to infect the respiratory tract mainly from the trachea to the lungs, causing pneumonia as a causative bacterium of parrot disease, and Chlamydophila felis (cat chlamydia) Infection has also been reported with examples of human infectious diseases such as infecting the mucous membrane of human eyes and causing conjunctivitis, and countermeasures against chlamydia as infection-causing bacteria are urgently needed.

  There are two major problems in dealing with Chlamydia infection. The first is the mildness of symptoms appearing on the surface. Infections caused by the sexually transmitted disease Chlamydia (obvious sexually transmitted infection) and infections caused by Chlamydia pneumonia are characterized by symptoms that usually resemble those of a common cold, and severe symptoms that may require treatment There is a problem that infection and symptoms are secretly advanced.

  The second problem is that when symptoms that require treatment appear, the antibiotics used in the treatment are superficially effective and are understood as if the disease was perceived. is there. Chlamydia infections are concealed by mild superficial symptoms, and as a result of long-term latent / persistent infections, sexually transmitted diseases chlamydia cause pelvic infections and infertility, and pneumonia chlamydia develops into lung cancer and arteriosclerosis There is a danger. As a strategy for Chlamydia infection, superficial treatment only when it worsens, such as treatment with antibiotics or antibodies, is insufficient, and it is necessary to take prevention and prevent persistent infection.

  Currently, development of vaccines against sexually transmitted diseases, Chlamydia pneumoniae and Chlamydia pneumoniae is also underway worldwide. Vaccines against animal chlamydia have been developed and are already on the market. Unfortunately, however, no vaccine has been developed that is effective against chlamydial infections. Moreover, in the vaccine using the whole microbial cell, the possibility that the heat shock protein contained in the microbial cell worsens the inflammatory reaction, arteriosclerosis, etc. by Chlamydia infection is reported. In other words, the strategy against Chlamydia infection includes the development of safer and more effective vaccines, and the establishment of treatment methods and constitutional improvements that can eliminate “potentially present Chlamydia” based on the characteristics of Chlamydia infection. It is essential.

Patent No. 3402304 Method for producing cat chlamydia vaccine Patent application title: Chlamydia Infectious Disease Treatment Agent JP-A-2001-288114 Autoimmune disease therapeutic agent Patent No. 3402304 Method for producing cat chlamydia vaccine

Rottenberg ME G-RA et al. (2002) Curr. Opin. Immunol. 14: 444-451 Belland RJ et al. (2003) Proc. Natl. Acad. Sci. U. S. A. 100: 15971-15976 Carlson LL. et al. (1989) Endocrinology 25: Jun. 70 Chan AS et al. (2002) Cell Signal. 14: 249-257 Shirai M HH et al. (2000) Nucleic Acids Res. 28: Apr. 11 Matsushima H et al. (2002) J. Org. Vet. Med. Sci. 64: 215-219

  In view of the above-mentioned present situation, the present invention aims to provide substances that effectively suppress the infection or proliferation of Chlamydia bacteria and methods for using these substances.

The present inventors are elucidating the pathogenicity of Chlamydia bacteria by genome analysis and information science comparative analysis. From the analysis, it has been clarified that in Chlamydia bacteria, there is a large “fluctuation” including the presence or absence of the tryptophan synthase gene group (FIG. 5). Focusing on this point, it was found that tryptophan metabolism intermediates, particularly melatonin and serotonin, have an effect of inhibiting chlamydia infection and proliferation while using tryptophan metabolism inhibitors in chlamydia infection experiments and the like. Melatonin and serotonin are known as neurotransmitters, but there are no similar reports on the effects of inhibiting chlamydial infection and proliferation in the well-known medical and biology-related literature. It was considered to be an action.

That is, the aspects of the present invention are as follows.

The first aspect of the present invention is an oral or injectable pharmaceutical composition for suppressing chlamydia infection or proliferation, characterized by comprising at least one drug selected from 5-hydroxytryptophan, melatonin and serotonin as an active ingredient It is a thing.

A second aspect of the present invention is a pharmaceutical composition, wherein the effective concentration of the drug in the first embodiment is in the range of 500μM of 5 [mu] M.

According to a third aspect of the present invention, among the active ingredients of the drug in the first aspect or the second aspect , 5-hydroxytryptophan is within a range of 50 μM to 500 μM, and serotonin is within a range of 50 μM to 100 μM. Melatonin is a pharmaceutical composition characterized by being in the range of 5 μM to 500 μM.

According to a fourth aspect of the present invention, in the first to third aspects, the target chlamydia is at least one of chlamydia trachomatis, chlamydia pneumoniae, chlamydia sitashi, chlamydia ferris. A pharmaceutical composition characterized by the above.

According to a fifth aspect of the present invention, in the first to fourth aspects, the chlamydial infection or growth suppression is an infection or proliferation of chlamydial infestation in at least one of the genital, lung, artery and trachea. It is a pharmaceutical composition characterized by suppression.

By using the pharmaceutical composition of the present invention , it is possible to produce a drug that is effective against Chlamydia spp. That is a causative bacterium of chronic infections and that is safe for the human body.

The pharmaceutical compositions of the present invention is relatively easy to infection Chlamydia bacteria, and can be produced safely, inexpensively preventable functional food and that Do.

The following describes the best mode for carrying out the present invention. A first aspect of the present invention is a tryptophan metabolic intermediate product , 5-hydroxytryptophan, melatonin, or serotonin (hereinafter referred to as “these”), characterized by suppressing the infection or growth of Chlamydia bacteria . A pharmacologically acceptable salt, ester or derivative has the same effect, so the description is omitted. An oral or injectable pharmaceutical composition comprising as an active ingredient is provided. As described above, although these materials are known, it is not known to have "the infection or growth inhibitory effect against Chlamydia bacteria" them as oral or injectable pharmaceutical as an active ingredient.

In order to obtain the effect of suppression particularly preferably, a pharmaceutical composition in which the concentration of 5-hydroxytryptophan, serotonin, and melatonin is in the range of 2 to 10 times the concentration in the body, or 5-hydroxytryptophan, serotonin The concentration of melatonin is in the range of 5 μM to 500 μM, specifically in the range of 50 μM to 500 μM for 5-hydroxytryptophan, in the range of 50 μM to 100 μM for serotonin, and in the range of 5 μM to 500 μM for melatonin. A pharmaceutical composition is disclosed. As described in the examples below, the present inventors have revealed that three substances of 5-hydroxytryptophan, serotonin, and melatonin have an inhibitory effect on chlamydia infection, so a pharmaceutical composition using this property is presented. Is done.

  Fig. 1 shows the tryptophan metabolic pathway in human cells, showing the effect on Chlamydia bacteria, and in addition to melatonin and serotonin synthesized in the human body, these are chemically modified substances that have an adverse effect on human cells. Substances that do not exert can also be used as pharmaceutical compositions. Both melatonin and serotonin are synthesized from tryptophan in the human body, but the above effects on Chlamydia spp. Cannot be expected at the concentration in the body. For example, it shows effects on human cells infected with Chlamydia spp. In the range of 2 to 10 times the concentration in the body is desirable. This concentration has a sufficient effect on Chlamydia bacteria and does not adversely affect human cells.

Examples of the target Chlamydia include at least one of Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia sitasi and Chlamydia ferris . These chlamydia are common in that they can infect human cells and cause disease, but the main parasites and pathological conditions differ depending on the species.For example, Chlamydia trachomatis affects the reproductive organs, Chlamydia Pneumoniae is known to infect inflammatory sites in the lungs and arteries, chlamydial weasel to airways from the trachea to lungs, and chlamydia ferris to the eye mucosa. It is necessary to administer the drug in an appropriate form for these individual infections, and by limiting the target bacteria, it is possible to develop a drug that can be expected to have a higher effect.

For example, pharmaceutical compositions of the present invention, characterized in that selected a drug composition that to suppress the infection or growth of Chlamydia bacteria, because the drug is any one of an oral agent, or injection drug A pharmaceutical composition is provided. As described above, Chlamydia spp. Have different main infection sites and symptoms for each species, and it is desirable that the mode of the drug corresponding to the infection site be adapted to the symptoms. For example, for a Chlamydia trachomatis infection oral agent, oral agent or injectable drugs against Chlamydia pneumoniae infection and Chlamydia familiarly infection, etc., dosage forms of drugs according to individual infection preferred. It is also within the scope of the present invention to create a drug having a higher effect by changing the concentration of the active ingredient or changing the combination of the drug in the individual forms of the bacteria. der Ru.

The meanings of the abbreviations used in this example are as follows. IFN-γ; interferon-γ, IFU; inclusion formation unit, FCS; physical calf serum, MOI; notification of infection, EB; elementary body, identi- ede3, ID3; : 5'-cyclic monophosphate.

HEp-2 cells (ATCC Ccl-23) were used as host cells for chlamydia infection experiments, in HEp-2 culture medium (Dulbecco's modified Eagle medium, 10% heat-inactivated FCS, 50 μg / ml gentamicin) at 37 ° C. Culturing was performed under a condition of carbon concentration of 5%. Examples of Chlamydia strains include C.I. pneumoniae J138 strain (Pneumonia chlamydia), C.I. felis Fe / C-56 strain (cat chlamydia), C.I. trachomatis sever D strain (sexually transmitted disease Chlamydia) was used. In the experiment, Escherichia coli, Staphylococcus aureus, Bacillus subtilis E192, Streptococcus pneumoniae, Pseudomonas aeruginosa PAO1, Helicobacter CP34 were used as other bacteria. The procedure of Chlamydia infection experiment is as follows. 2 × 10 4 (in 0.1 ml) HEp-2 cells are dispensed into a 96-well plate and cultured for 24 hours. Chlamydia EB is added here so that it may become 0.2 MOI, and it centrifuges at 22 degreeC and 700 xg for 60 minutes, and sets it for 30 minutes. These cells are transferred to a post-infusion medium (Dulbecco's modified Eagle medium, 5% heat-inactivated FCS, 50 μg / ml gentamicin, 1 μg / ml cycloheximide), and cultured at CO _{2 for} 5 hours at 36 ° C. for 5 hours. A schedule of drug treatment in the experiment is shown in FIG. Chlamydia infection rate was measured by the following method. Infected host cells were fixed with methanol for 30 minutes, FITC-labeled Chlamydia bacteria-specific antibody was reacted for 1 hour (37 ° C.), and then the host cell nucleus was stained with DAPI (0.1 μg / ml) for 10 minutes. Host cells and Chlamydia inclusion bodies (diameter 1.0 μm or more) were counted using a fluorescence microscope. The infection rate was calculated from the average value of three independent experiments. The effect of melatonin on bacteria other than the above chlamydia was examined by adding melatonin or gentamicin to each medium, culturing at 37 ° C. for 8 hours (24 hours for H. pylori), and then measuring 600 nm OD.

That put the infection experiments Chlamydia pneumoniae J138 strain in vitro using a human-derived HEp-2 cells, 5-hydroxytryptophan, serotonin, melatonin were significantly Chlamydia growth inhibitory effect compared to the control (FIG. 1). In order to compare with the known chlamydial growth inhibitory effect of IFN-γ (which causes tryptophan deficiency, the effect is counteracted by the addition of tryptophan), when tryptophan addition experiment was conducted, IFN-γ In contrast, addition of tryptophan did not counteract the growth inhibitory effect of chlamydia (FIG. 2), and it was not observed that chlamydial infection led to persistent infection. Furthermore, it was found that melatonin and serotonin have an inhibitory effect on feline chlamydia with almost complete set of tryptophan synthesis gene, partly sexually transmitted disease chlamydia and completely lacking pneumoniae chlamydia. (Figure 3). On the other hand, no inhibitory effect was observed on the 10 types of human resident bacteria even when the pharmaceutical composition of the present invention was administered at a high concentration.

  In order to clarify the effects of melatonin and serotonin on chlamydia infection, this cell is pretreated with these substances to determine whether this effect is on chlamydia or on host cells, and subsequent infection The effect on As a result, when host cells were pretreated with melatonin or serotonin, an infection inhibitory effect was observed, but pretreatment on chlamydia cells had little effect on infection (FIG. 4). This result indicates that the effects of melatonin and serotonin are on host cells. Regarding melatonin and serotonin, it is known that there are receptors involved in uptake and signal transduction in various human tissue cells, and many of these receptors are receptors that bind G protein. is there. Therefore, pertussis toxin, which is an inhibitor of G protein, was used to examine the influence of melatonin and serotonin on the inhibitory effect on chlamydia infection. As a result, for serotonin, the pertussis toxin treatment counteracted the infection inhibitory effect, while melatonin did not affect the infection inhibition with melatonin (FIG. 4). This indicates that the host G protein is involved in the chlamydia inhibitory effect of serotonin but not the melatonin effect.

Figure 2 shows the effect of tryptophan and its metabolic intermediates on chlamydia infection in in vitro conditions. A: A schematic diagram of a tryptophan metabolic pathway comprising a kynurenine pathway and a serotonin pathway is shown. B: Represents time settings for chlamydia infection and drug treatment under in vitro conditions in this study. In most experiments, drug treatment was performed 24 hours pre-infection and 48 hours post-infection. C: Shows the effect of tryptophan and its metabolic intermediates on chlamydia infection. The symbols A to G on the horizontal axis correspond to the symbols on the right shoulder of the metabolite in FIG. 1A (A = L-tryptophan, B = 5-hydroxytryptophan, C = kynurenine, D = serotonin, E = melatinin, respectively. , F = 5-hydroxytryptophanol, G = 5-methoxyindole acid acid), the numbers are C.I. for human HEp-2 cells. It represents the concentration (50,200 μM) of each substance at the time of infection experiment of Pneumoniae J138 strain. None of A to G showed toxicity to host cells under the conditions of this experiment. The vertical axis of the graph shows the relative value of the infection rate with respect to the control (no treatment) (control = 100), the graph shows the average value in three repeated experiments, and the attached bar shows ± 1SD. Those with a significant difference from the control were marked with * (t test, p <0.01). These are all the drugs of the present invention. The effects of melatonin and interferon-γ (IFN-γ) on Chlamydia infection were compared. A: Shows the effect of each substance on Chlamydia infection. + And − represent the presence or absence of melatonin (100 μM), IFN-γ (1.0 ng / ml) and tryptophan (200 μg / ml), respectively. The vertical axis is the relative value of the infection rate relative to the control (left end, 100), each graph shows the average value of three repeated experiments, and the attached bar shows ± 1 SD. B: Inclusion bodies in Chlamydia infection were visualized with FITC-labeled Chlamydia specific antibodies (white squares in Inclusion body row). Each bar in the lower right of the photo is 10 μm. C: Size distribution (radius, horizontal axis) of inclusion bodies in each of melatonin treatment, IFN-γ treatment, and control. The comparison of melatonin sensitivity by Chlamydia species is shown. C. pneumoniae, C.I. felis, C.I. Each trachomatis was treated with melatonin at 25 μM, 50 μM, and 100 μM, and compared with IFN-γ treatment. The vertical axis of the graph indicates the relative infection rate (control = 100), and the horizontal axis indicates the presence or absence of treatment. C. to HE-p2 cells. We searched for melatonin and serotonin targets in pneumoniae infection. A: HE-p2 cells, C.I. Each of pneumoniae J138 strain was treated with melatonin / serotonin alone, and the influence on subsequent infection was examined. Chlamydia cells were treated at the concentration of 200 μM in the same solution as the HEp-2 cell culture solution 90 minutes before infection. * Indicates that there was a significant difference between control and treatment (Student t test, p <0.05). B: Pertussis toxin (pertussis toxin, 20 ng / ml), which is a G protein inhibitor, was added to each of melatonin treatment and seratonin treatment for 48 hours, and the effect was examined. The schematic diagram of the tryptophan synthesis pathway in Chlamydia bacteria and a host cell is shown. As genes involved in tryptophan biosynthesis in Chlamydia bacteria, kynU, kprS, trpD, trpF, trpC, trpF, trpC, trpA, trpB work at each stage of the synthetic pathway. C. Infecting cats Felis has all of these gene sets, but C. infects humans. trachomatis has only an incomplete gene set. no pneumoniae. It is known that administration of interferon-γ against Chlamydia bacteria causes tryptophan deficiency in the kynurenine pathway and causes chlamydia inhibition.

Claims (5)

An oral or injectable pharmaceutical composition for suppressing chlamydia infection or proliferation, comprising at least one drug selected from 5-hydroxytryptophan, melatonin and serotonin as an active ingredient. The pharmaceutical composition of claim 1, wherein the effective concentration of the drug is in the range of 500μM of 5 [mu] M. Among the active ingredients of the drug, 5-hydroxytryptophan is within a range of 50 μM to 500 μM, serotonin is within a range of 50 μM to 100 μM, and melatonin is within a range of 5 μM to 500 μM. Item 3. A pharmaceutical composition according to Item 1 or 2 . 4. The pharmaceutical composition according to any one of claims 1 to 3 , wherein the chlamydia is at least one of chlamydia trachomatis, chlamydia pneumoniae, chlamydia sitasi, chlamydia ferris. The said chlamydia infection or growth suppression is the infection with respect to the chlamydial infestation in at least one among a genital organ, a lung, an artery, and a trachea, or growth suppression, The any one of Claims 1 thru | or 4 characterized by the above-mentioned. Pharmaceutical composition.

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