KR101734323B1 - Composition of prevention and treatment for tuberculosis comprising stimulator of expression or activity of prostate apoptosis response-4 - Google Patents
Composition of prevention and treatment for tuberculosis comprising stimulator of expression or activity of prostate apoptosis response-4 Download PDFInfo
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Abstract
The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of tuberculosis comprising Par-4 and GRP78 expression or activity promoter as an active ingredient. In the case of increasing the expression of Par-4 (prostate apoptosis response-4) or GRP78 (78 kDa glucose-regulated protein) of the present invention, it is possible to prevent tuberculosis from being killed, Positive effects can be expected. In addition, treatment effects of multi-drug resistant (MDR) and extensively drug-resistant (XDR), one of the difficulties of treating tuberculosis in recent years, can be expected. It is possible to shorten the period for taking the medicines and to relieve the suffering of the patient due to the long time taking of the medicines and to prevent the failure and the recurrence of the treatment due to the irregular use of the medicines of the patients.
Description
The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of tuberculosis comprising Par-4 and GRP78 expression or activity promoter as an active ingredient.
Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis. It is not only a major disease in developing countries, but also increasingly serious in the developed countries of the world. About 8 million patients Approximately 3 million patients die. Although tuberculosis can be symptom free for a considerable period of time when infected, it most commonly results in acute inflammation of the lungs, leading to fever and unproductive cough, which, if not treated, will usually lead to serious complications and death.
The importance of these Mycobacterium tuberculosis is increasing because of the incidence of Mycobacterium tuberculosis strains that are resistant to global epidemics of HIV and several drugs. Investigations of antibiotic-resistant Mycobacterium tuberculosis in 35 countries in the last five years have shown that 36% of patients have resistance to one or more drugs and that two or more of them, including isoniazid (INH) and rifampin (RMP) Multidrug-resistant Mycobacterium tuberculosis (MDR-TB), which is resistant to antibiotics, is at a serious level of approximately 13%. Even in patients without a history of treatment, the resistance to at least one drug is 9.9%. In this regard, tuberculosis, especially drug-resistant and multidrug-resistant tuberculosis, not only leads to an increase in the cost of treatment, but also lowers treatment efficiency and poses a serious threat to the development of incurable tuberculosis. Conventional tuberculosis treatments usually require long-term treatment for 1 to 2 years. When 1-2 anti-tuberculosis agents are used, they are rapidly tolerated. Therefore, it is recommended to administer 3-4 drugs when treating with anti-tuberculosis agents. However, when given over a long period of time, it causes a side effect such as cirrhosis and jaundice. In addition, for the treatment of multidrug-resistant tuberculosis, there are problems that are relatively less effective, more side effects are caused, and high-cost secondary anti-tuberculosis drugs should be taken. Therefore, in order to improve the treatment efficiency of such multidrug-resistant tuberculosis and to eliminate the tuberculosis, there is a need to develop a new drug that can treat up to latent tuberculosis, is more effective, has few side effects, and can be used for a short period of time . Thus, there is a desperate need for the development of a safe and effective anti - tuberculosis agent in the human body, but the currently developed therapies are not effective in the treatment of tuberculosis.
In addition, Par-4 (prostate apoptosis response-4) is a tumor suppressor protein that exists in various parts of the cell and has a Leucine Zipper (LZ) domain and a Selective for apoptosis of cancer cells ) Are known to have. In addition, Par-4 secretes out of the cell after binding with GRP78, which is increased in the case of endoplasmic reticulum stress (ER stress), and induces apoptosis by activating caspase-8.
GRP78 is one of the proteins of the heat shock protein family. It is present in the endoplasmic reticulum and is involved in the folding of the protein. It increases the unfolded protein and the misfolded protein in the endoplasmic reticulum Or when the intracellular calcium ion homeostasis is broken down and stress is generated in the cells, it is known that GRP78 is increased.
Recently, Par-4 has been reported to be highly expressed in cancer cells, and it has been reported to control the tumor. Drugs that increase the expression of Par-4 have been used in cancer cells and have been used as anticancer drugs. It is used in various ways to treat cancer related to the same hormone. However, the fact that Par-4 and GRP78 are involved in the prevention and treatment of tuberculosis has not been reported at all.
Accordingly, the inventors of the present invention found that when the expression of Par-4 (prostate apoptosis response-4) and GRP78 (78 kDa glucose-regulated protein) was decreased, the increase of Mycobacterium tuberculosis in the macrophages and the increase of the expression of Par- , Confirming that the tuberculosis bacillus is decreased, and confirming that it is effective for the prevention or treatment of tuberculosis, thereby completing the present invention.
It is an object of the present invention to provide a pharmaceutical composition for preventing or treating tuberculosis comprising, as an active ingredient, an expression-promoting agent or an activity promoting agent of any one or more of Par-4 (prostate apoptosis response-4) and GRP78 (78 kDa glucose- .
Another object of the present invention is to provide a method for the prophylaxis or treatment of tuberculosis using the promoter or activator of Par-4 (prostate apoptosis response-4) and GRP78 (78 kDa glucose-regulated protein).
Another object of the present invention is to provide a method for screening candidate substances for preventing or treating tuberculosis, which comprises measuring the degree of expression of Par-4 or GRP78 gene or the activity of a protein encoded by the gene.
The present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising, as an active ingredient, an expression promoter or an activity promoter of any one or more of Par-4 (prostate apoptosis response-4) and GRP78 (78 kDa glucose-regulated protein).
Hereinafter, the present invention will be described in detail.
In the present invention, "expression promoter or activity promoter" of any one or more of Par-4 (prostate apoptosis response-4) and GRP78 (78 kDa glucose- regulated protein) refers to Par- -4, or GRP78 in a mammal. Such materials include single compounds such as organic or inorganic compounds, biopolymer compounds such as peptides, proteins, nucleic acids, carbohydrates and lipids, and complexes of multiple compounds. The mechanism by which the substance promotes the expression or activity of the Par-4 or GRP78 is not particularly limited. For example, a substance may act as a mechanism to increase gene expression, such as transcription, translation, or to convert an inactive form to an active form. Preferably, the substance that promotes the expression or activity of Par-4 or GRP78 is a biopolymer compound such as a peptide, protein, nucleic acid, carbohydrate and lipid. For the known Par-4 or GRP78 nucleic acid and protein sequences, those skilled in the art will appreciate that a single compound such as an organic or inorganic compound acting as a promoter, a biopolymer compound such as a protein, nucleic acid, carbohydrate and lipid, And can be manufactured or screened using techniques in the art.
The expression or activity promoter of Par-4 or GRP78 of the present invention may be provided in the form of a vector capable of expressing Par-4 or GRP78 in cells for use in gene therapy and the like. Accordingly, the present invention relates to a composition for preventing and treating tuberculosis comprising a nucleic acid encoding Par-4 or GRP78, preferably a recombinant vector containing the nucleic acid as an active ingredient.
The nucleic acid sequence encoding Par-4 or GRP78 can be mutated by substitution, deletion, insertion, or a combination thereof, as long as one or more nucleic acid bases encode a protein having equivalent activity. The sequence of such a nucleic acid molecule may be short or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
The vector of the present invention includes, but is not limited to, liposomes, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Examples of preferred viral vectors in the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, alpha virus). The recombinant vector of the present invention may contain a nucleic acid encoding Par-4 or GRP78 and a regulatory sequence for transcription or translation thereof. A particularly important regulatory sequence is the regulation of transcription initiation, such as promoters and enhancers. It may also contain regulatory sequences consisting of initiation codon, termination codon, polyadenylation signal, Kozak, signal sequence for enhancer, membrane targeting and secretion, IRES (Internal Ribosome Entry Site), and the like. Such a regulatory sequence and a nucleic acid encoding Par-4 or GRP78 should be operably linked.
The term " operably linked " as used herein means that the linkage between nucleic acid sequences is functionally related. When any nucleic acid sequence is operably linked, any nucleic acid sequence is located so as to be functionally related to another nucleic acid sequence. In the present invention, it is said that when any transcriptional regulatory sequence affects the transcription of a nucleic acid molecule encoding Par-4 or GRP7, the transcriptional control sequence is operably linked to the nucleic acid molecule. In the present invention, the treatment includes inhibiting or preventing tuberculosis, or reducing, alleviating, reversing, and inhibiting the progress of tuberculosis.
The pharmaceutically acceptable carriers to be included in the composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
The pharmaceutical composition of the present invention can be administered through a route commonly used in gene therapy, and parenteral administration is preferred, and for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration Lt; / RTI >
The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and responsiveness of the patient And the ordinarily skilled physician can easily determine and prescribe dosages effective for the desired treatment. In one embodiment, the pharmaceutical composition of the present invention may be administered intravenously 1 to 2 times with 1 x 10 8 to 1 x 10 11 pfu of recombinant adenovirus.
The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing agents or stabilizers.
The pharmaceutical compositions of the present invention can be lyophilized to increase stability at room temperature, reduce the need for costly cold storage, and prolong shelf-life. The lyophilization process may be carried out in successive stages of freezing, primary drying and secondary drying. The secondary drying process after freezing the composition is to lower the pressure and heat it for sublimation of water vapor. The secondary drying step is to evaporate the residual moisture absorbed from the dried material.
In addition, the present invention provides a method of preventing or treating tuberculosis, comprising administering to a subject an expression promoter or an activity promoter of Par-4 or GRP78.
The method for preventing or treating tuberculosis of the present invention comprises administering an effective amount of an expression promoter or an activity promoting agent of Par-4 or GRP78 gene of the present invention to an individual. The specific therapeutically effective amount for a particular individual will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned factors. In the present invention, the subject is applicable to any mammal, including humans and primates as well as livestock such as cows, pigs, sheep, horses, dogs and cats.
Further, the present invention provides a method for producing a biological sample, comprising the steps of: 1) treating a biological sample with a test substance; 2) measuring the degree of expression of the Par-4 or GRP78 gene or the degree of activity of the protein encoded by the gene in the biological sample; And 3) selecting a test substance whose expression level of Par-4 or GRP78 gene or activity level of Par-4 or GRP78 protein is increased compared to a control group not treated with the test substance. And a screening method for the candidate substance for screening.
In the above method, the biological sample of step 1) includes blood, urine, saliva and tissue of an individual, and preferably a cell line. The cell line includes all cell lines, preferably human immune cells, more preferably macrophages. In the above method, the degree of expression of the gene in step 2) may be determined by a method such as RT-PCR (reverse transcription polymerase chain reaction), Northern blot, cDNA microarray hybridization, and in situ hybridization And any method of measuring the amount of a gene known to a person skilled in the art can be used, but is not limited thereto.
In the above method, the degree of activity of the protein in step 2) may be determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry ), And the like. Any method of measuring the amount of a protein known to a person skilled in the art can be used, but is not limited thereto.
In the case of increasing the expression or activity of Par-4 (prostate apoptosis response-4) or GRP78 (78 kDa glucose-regulated protein) of the present invention, it is possible to kill tubercle bacilli as well as control the proliferation of tubercle bacilli, A positive effect on the treatment can be expected. In addition, treatment effects of multi-drug resistant (MDR) and extensively drug-resistant (XDR), one of the difficulties of treating tuberculosis in recent years, can be expected. It is possible to shorten the period for taking the medicines and to relieve the suffering of the patient due to the long time taking of the medicines and to prevent the failure and the recurrence of the treatment due to the irregular use of the medicines of the patients.
1 is a graph showing the expression levels of Par-4 and GRP78 in macrophage infections of Mycobacterium tuberculosis H37Ra (protein level: western blot)
FIG. 2 is a graph showing the expression level of Par-4 and GRP78 when infected with macrophage of Mycobacterium tuberculosis H37Ra (RNA level; Reverse Transcription Polymerase Chain Reaction).
FIG. 3 is a graph showing the expression levels of Par-4 and GRP78 as the ratio of mycobacterial cells per macrophage increases.
Fig. 4 shows the number of Mycobacterium tuberculosis in macrophages when the expression of Par-4 and GRP78 was decreased. Fig.
FIG. 5 shows the number of Mycobacterium tuberculosis in macrophages overexpressing Par-4. FIG.
Hereinafter, the present invention will be described in detail with reference to examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited by the examples.
[ Example 1] After infection with M. tuberculosis in macrophages, Par-4 and GRP78 Identification of changes in expression
In order to confirm the expression and function of Par-4 and GRP78 in the intracellular infection of Mycobacterium tuberculosis, the expression of Par-4 and GRP78 in the mouse-derived macrophage Raw264.7 cells was infected with Mycobacterium tuberculosis H37Ra And the expression level of Par-4 and GRP78 was measured by the ratio of cells per cell and time. The expression level of Par-4 and GRP78 protein in the macrophage at a ratio of 5 cells per cell was determined by Western blotting according to the incubation time in the case of Mycobacterium tuberculosis infection, The results of RT-PCR analysis of the gene expression of Par-4 and GRP78 in Mycobacterium tuberculosis infection are shown in Fig. 2. The ratio of 1, 5, 4 and GRP78 using Western blotting technique. The results are shown in Fig.
As shown in FIG. 1, it was confirmed that Par-4 was expressed only at 24 hours of culture and decreased again at 48 hours, and GRP78 expression was also increased with an increase in incubation time.
As shown in Fig. 2, the expression of Par-4 mRNA significantly increased after 0.5 hours of infection, then decreased gradually, and the expression of mRNA of GRP78 increased in proportion to time.
As shown in FIG. 3, it was confirmed that the expression of Par-4 and GRP78 was increased as the number of cells per cell increased.
These results indicate that cells infected with Mycobacterium tuberculosis in macrophages increase the expression of Par-4 and GRP78 in one way to counteract M. tuberculosis.
[ Example 2] Par-4 and GRP78 Identification of Mycobacterium tuberculosis Growth Effect by Decreased Expression
To determine the effect of Par-4 and GRP78 expression on the growth of Mycobacterium tuberculosis in intracellular infections of Mycobacterium tuberculosis, the number of Mycobacterium tuberculosis in macrophages was measured when the expression of Par-4 and GRP78 decreased.
More specifically, transfection of Par-4 siRNA, GRP78 siRNA and negative control siRNA into macrophages was performed using GenMute at a concentration of 150 nM and incubated in an incubator under CO 2 at 37 ° C for 5 hours Lt; / RTI > Then, macrophages were cultured in complete medium containing 5% fetal bovine serum and no antibiotics for 24 hours, infected with Mycobacterium tuberculosis for 3 hours, and then washed with extracellular mycobacteria. Thereafter, the cells were cultured in a complete medium for 24 hours. Fig. 4 shows the results of measuring the number of tubercle bacilli surviving in macrophages after 0 to 24 hours from infecting the transfected cells with Mycobacterium tuberculosis.
As shown in FIG. 4, in comparison with the negative control, the number of Mycobacterium tuberculosis was significantly increased due to a decrease in the expression of Par-4, and the number of Mycobacterium tuberculosis was increased when the expression of GRP78 was decreased.
[ Example 3] Decrease in the number of mycobacteria in the macrophages with overexpression of Par-4
To determine the effect of increasing Par-4 expression on the growth of Mycobacterium tuberculosis in intracellular infections of Mycobacterium tuberculosis, the number of Mycobacterium tuberculosis cells in macrophages overexpressing Par-4 was measured.
More specifically, the overexpression of Par-4 and GRP78 was induced by adding negative control pcDNA3.1 and pcDNA3.1-Par-4 to Raw 264.7 cells using Lipofectamine 3000 at a concentration of 1 [mu] g / Lt; / RTI > After incubation for 5 hours at 37 ° C in a 5% CO 2 incubator, macrophages were cultured in complete medium containing 5% fetal bovine serum and no antibiotics for 24 hours. Thereafter, the cells were infected with Mycobacterium tuberculosis for 3 hours, and the extracellular mycobacteria was washed. Then, the cells were cultured in complete medium for 24 hours. Fig. 5 shows the results of measuring the number of tubercle bacilli surviving in the cells after passage of 0 to 24 hours after infecting the macrophages with Par-4 overexpressing them.
As shown in FIG. 5, when the expression of Par-4 was increased, the number of mycobacterial cells in the cells was significantly reduced as compared with the control group. Thus, it can be seen that when Par-4 is overexpressed, tuberculosis can be prevented or treated.
Claims (8)
2) measuring the degree of expression of the Par-4 gene or the degree of activity of the protein encoded by the gene in the biological sample; And
3) selecting a test substance whose expression level of Par-4 gene or activity level of Par-4 protein is increased compared to a control group not treated with the test substance;
A method for screening candidate substances for the prevention or treatment of tuberculosis.
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