TW201704257A - Use of polypeptide for the manufacture of pharmaceutical composites for the treatment and/or prevention of diseases associated with target points having target points consisting of varuious genes, proliferators, and message transport route for the treatment of different kinds of diseases - Google Patents

Abstract

The present invention discloses the use of polypeptide for the manufacture of pharmaceutical composites for the treatment and/or prevention of diseases associated with target points, wherein the polypeptide comprises a sequence encoded as SEQ ID No.1, and further the targets are selected from the groups consisting of proliferator-activated receptor, nuclear factor-Kappa B and Sirt gene.

Description

Use of a multi-peptide for the manufacture of a pharmaceutical composition for treating or/and preventing a target-related disease

The present invention relates to a use of a multi-peptide to produce a pharmaceutical composition for regulating a multi-site, multi-gene, multi-target, wherein the multi-peptide is encoded with the sequence encoded as SEQ ID No. 1 or The homologous amino acid sequence derived by the substitution, deletion, or addition of one or more amino acids.

According to the advancement of biomedical technology, many studies have revealed the effects of different genes, different growth factors, different message transmission pathways, and different targets on disease occurrence and progression, and researchers have developed a relationship between target and disease. It can directly act as a target drug for a specific target. Currently, the target drug is most commonly used for the treatment of cancer diseases, even if the target drug directly acts on the oncogenic gene that induces cancer, and achieves the effect of treating cancer. For example, lung cancer patients with mutations to the epithelial factor receptor can be administered to target drugs such as Iressa or Tarceva for more effective treatment and to reduce the side effects of traditional drugs on individuals. .

In addition, pemetrexed is a new generation of anti-metabolic anticancer drug successfully developed by Eli Lilly. It is a multi-target anti-cancer drug, and its target is the key enzymes in the folate metabolism pathway. Since this pathway can significantly affect the DNA synthesis and cancer growth of cancer cells, it has been confirmed that the inhibitory effect of pemetrexed on cancer is not only significant, but also may have therapeutic effects on various cancers. The US Food and Drug Administration (FDA) has been approved for the treatment of malignant pleural mesothelioma and advanced non-small cell lung cancer. Furthermore, a large-scale international multi-center phase III clinical study found that the combination of pemetrexed chemotherapy with cisplatin can further improve the efficacy of malignant pleural mesothelioma that cannot be surgically removed. Extend the patient's survival time. Furthermore, another large-scale phase III clinical study found that patients with advanced non-small cell lung cancer were treated with pemetrexed after failure of first-line chemotherapy, and the efficacy was not lower than that of commonly used docetaxel. And some side effects are even lower than the latter. In addition, pemetrexed has shown certain effects in the treatment of various malignant tumors, such as gastric cancer, breast cancer, pancreatic cancer, etc. Even in lung cancer and mesothelioma, the range of patients for which it is applied is also expanding.

Another study found that the CDA-2 human urine preparation has the ability to regulate multiple genes. In particular, the CDA-2 human urine preparation system can solve the abnormal state of cancer cell methyltransferase, so that cancer cells do not continue to divide, and induce cancer cells to undergo terminal differentiation or apoptosis, thereby achieving the purpose of treating cancer. . It can be seen from the cell test that CDA-2 human urine preparation can induce the apoptosis of blood cancer cells HL-60 and NB4 and Hep3B cells, and can reduce the activity of caspase 3 (Caspase 3). In addition, from the results of animal experiments, it can be seen that CDA-2 human urine preparation has the ability to down-regulate genes related to cell proliferation, including TGF-2, PCNA, c-myc, c-jin, c-fos, N-ras and so on. CDA-2 human urine preparations can cause cancer cells to stay in the first phase of the cell growth phase by up-regulating genes that inhibit the cell cycle, such as P16, P21 and P27, and down-regulating the cyclin D1 cell cycle. G1arrest).

Furthermore, CDA-2 human urine preparations can inhibit the expression of vascular proliferative factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and inhibitory antibodies. The performance of drug factors, such as Her-2/neu protein, can inhibit vascular proliferation and alter drug resistance. CDA-2 human urine preparations can inhibit metastasis by down-regulating and translating proteins involved, such as matrix metalloproteinase-9 (MMP-9), integrin beta 1 (lntegrin β 1). In addition, the CDA-2 human urinary system activates peroxisome proliferator-activated receptor γ (PPARγ), which causes cancer cells to return to normal cells and inhibit cancer.

Accordingly, the CDA-2 human urine preparation can act on different targets, and therefore, the CDA-2 human urine preparation has a wider range of indications than the conventional medicine, and can inhibit the growth and migration of cancer cells. The ability to reduce the side effects and achieve effective treatment of cancer, prolong the life of patients, and improve the quality of life of patients. It can be seen that when the pharmaceutical composition can act on different targets, or the target of its action can affect or regulate the progress of different diseases, it is possible to treat different diseases by administering the pharmaceutical composition to a specific target. efficacy.

In addition to the use of targeted drugs for the treatment of cancer, the indications for current targeted drugs still include non-cancer diseases. In other words, the target of targeted drugs is no longer limited to those associated with cancer diseases. Specifically, the Republic of China Patent No. I360576 "The use of resveratrol for the treatment of atypical teratoid/rhinomatous cell tumor caused by cancer stem cells" attenuates stem cell genes by inhibiting the expression of the Sirt1 gene or It is the expression of drug resistance genes, which helps to improve the effect of radiotherapy of tumor cells. Sirt1 protein can trigger fat breakdown and reduce fat content in mature fat cells, and it is confirmed in the study of skeletal muscle metabolism that Sirt1 activator such as resveratrol can activate the Sirt1 gene to deacetylate PCG-1α protein. It is activated to achieve the function of activating mitochondrial biosynthesis genes and regulating the genes involved in energy metabolism.

The Sirt1 gene is located on the 10th pair of chromosomes of human chromosomes, interspersed in the nucleus and cytoplasm, and is transcribed into a Sirt1 protein with a molecular weight of approximately 81.7 kDa. Sirt1 egg The white matter belongs to the third family of NDA+deacetylases, which can stimulate cell repair, resist inflammation, protect neurons, and resist apoptosis by regulating the mechanism of proteins, thereby improving health and prolonging life.

According to previous studies, Sirt1 protein binds directly to the gene promoter of uncoupling protein-2 (UCP2), inhibits the expression of uncoupling protein 2, and regulates insulin secretion and glycolipid metabolism. In the study of cultured hepatocytes, it was confirmed that Sirt1 protein can interact with FOXO1 in the nucleus to deacetylate FOXO1, which can promote the gluconeogenesis in the liver and provide energy for cell survival. In addition, peroxisome proliferator-activated receptor gamma ligand 1α (PPAR-γ Coactivator 1-α, hereinafter referred to as PCG-1α) is a peroxisome proliferator-activated receptor γ (PPAR-γ). The co-living compound is also one of the substrates of the Sirt1 protein. Under normal physiological conditions, Sirt1 protein interacts with PCG-1α, increasing the activity of PCG-1α, thereby improving the performance of liver gluconeogenesis genes and skeletal muscle fatty acid oxidation. Based on this, many target drugs currently use the Sirt1 gene as a target.

In addition, as in the Republic of China Patent No. I406668 "a pharmaceutical composition for inhibiting the growth of Helicobacter pylori and inhibiting the inflammatory response of Helicobacter pylori-induced gastric epithelial cells", it is possible to inhibit the activation of nuclear factor kappa B by Helicobacter pylori. It achieves the effect of inhibiting the inhibition of Helicobacter pylori-induced inflammation of gastric epithelial cells. Nuclear factor-κB (NF-κB) is a nuclear transcription factor, a dimeric protein composed of secondary molecules, wherein the sub-molecule contains p50, p65, p52, RelB, c-Rel. And other proteins. According to recent studies, nuclear factor kappa B is closely related to inflammatory response, apoptosis, cell necrosis, and cancer formation. In the cytoplasm, the nuclear factor-κB line binds to a negative regulatory protein (hereinafter referred to as IκB), and when the cell is stimulated by external stimulation, the negative regulatory protein is degraded by phosphorylation and loses its activity. At this time, nuclear factor kappa B enters the nucleus. The expression of the gene is activated by binding to the promoter of the regulated gene. For example, rats whose genes have been removed from IKK (IκBkinase) can prevent the release of NF-κB into the nucleus and reduce the post-traumatic inflammatory response after IKK can not inhibit IκB. Conversely, when a substance released by a foreign substance or cell inhibits IκB, such as lipopolysaccharide (LPS), nuclear factor kappa B is activated to enter the nucleus, resulting in the production of, for example, an inflammatory factor.

In summary, a variety of genes, growth factors, message transmission pathways, etc. can be used as targets for targeted drugs, such as Sirt1 protein or nuclear factor κ B. However, the technical features disclosed in the previous patent can only be used for A single disease has a therapeutic effect and cannot be used for multiple indications at the same time, increasing the cost of drug development. In order to avoid the lack of prior art, the inventors of the present invention achieve the regulation of physiological functions by acting on multi-site, multi-gene, multi-target peptides, for example, Sirt1 protein can be obtained by nuclear factor kappa B The sub-molecules (p65, p52) are deacetylated to avoid binding of nuclear factor-κB to genes involved in the inflammatory reaction in the nucleus, reducing the expression of inflammatory factors such as tumor necrosis factor alpha (TNF-α) and IL-1β, and inhibiting the inflammatory response. The company is committed to developing a pharmaceutical composition that can be used to improve or treat related diseases, not only to achieve multiple effects at the same time, but also to enhance the public interest and save development costs.

The main object of the present invention is to disclose the use of the amino acid sequence encoded by the amino acid sequence of SEQ ID No. 1 for the manufacture of a pharmaceutical composition.

In order to achieve the above object, embodiments of the present invention disclose the use of a multi-peptide for the manufacture of a pharmaceutical composition for treating or/and preventing a target-related disease, wherein the multi-win The peptide has a sequence encoding SEQ ID No. 1 or a homologous amino acid sequence derived by substitution, deletion, or addition of one or more amino acids, and the target is selected from the group consisting of proliferating factors A group consisting of a receptor, a nuclear factor kappa B, and a Sirt1 gene. .

Preferably, the amino acid sequence of the multi-peptide is encoded as the sequence of SEQ ID No. 1.

Preferably, the amino acid sequence of the multi-peptide is more than 90% similar to the sequence encoded as SEQ ID No. 1.

Preferably, the disease is muscular atrophy.

Preferably, the disease is a condition with an inflammatory response.

Preferably, the disease is associated with metabolic syndrome, wherein, in particular, the disease may be fatty liver or obesity.

Since the multi-peptide system of the present invention has the ability to regulate multi-gene transcription or multi-target expression, the pharmaceutical composition is administered to a body by administering an effective amount of the multi-peptide of the present invention. The composition system can act on at least one specific target to achieve the effect of multiple effects in the individual.

The first figure is the result of protein electrophoresis confirming the homologous protein of the multi-peptide homologous glycoprotein contained in the extract of Cucurbitaceae.

The second panel is the result of synthesizing the multi-peptide encoded by the amino acid sequence of the present invention encoded as SEQ ID No. 1 by an automated synthetic multi-peptide device.

The third figure will encode the amino acid sequence of the present invention produced by the recombinant bioproduction platform as The result of the multi-peptide of SEQ ID No. 1.

Figure 4A shows the results of cold-light enzyme activity of each group of mice observed by living images.

The fourth panel B is the statistical analysis of the cold light values of each group of mice.

The fifth figure is the measurement and analysis of the number of photons emitted by each organ of each group of mice in the luminescence image.

The sixth graph is the result of immunostaining analysis of the organs of each group of mice.

Figure 7A shows the results of H&E staining of liver tissues of each group of mice.

Figure 7B shows the results of oil red O staining of liver tissues of each group of mice.

Fig. 8A shows the results of immunohistochemical staining of liver tissues of mice of each group with 4-hydroxydecenoic acid antibody.

Figure 8B shows the results of immunohistochemical staining of liver tissues of mice of each group with malondialdehyde antibody.

The ninth panel is the appearance of the experimental group mice and the control group mice.

The tenth figure is the adipose tissue of the experimental group and the control group.

The eleventh figure shows the results of staining the adipose tissue of the experimental group and the control group.

The twelfth image shows the results of H&E staining of the muscle tissues of the experimental group and the control group.

The meaning of the technical and scientific terms used in the description and claims of the present invention are the same as those of ordinary skill in the art to which the present invention pertains. In case of conflict, the content of the present invention shall prevail.

The present invention discloses that the amino acid sequence is encoded as the multi-peptide of SEQ ID No. 1 or The use of its homologous amino acid sequence. Since the amino acid sequence encoded by the present invention encodes the multi-peptide of SEQ ID No. 1 or a homologous amino acid sequence thereof having the ability to regulate multi-gene transcription or multi-target expression, the multi-peptide or The homologous amino acid sequence has the following effects: one, applied to multiple parts of an individual, acting on a multi-gene target; second, treating an inflamed or inflammatory reaction; and third, inhibiting liver fat deposition Symptoms; Fourth, the effect of inhibiting the accumulation of fat; Fifth, the effect of preventing muscle atrophy; Sixth, reducing the complications of diabetes.

Accordingly, by administering an effective amount of the amino acid sequence encoding the polypeptide of SEQ ID No. 1 or a homologous amino acid sequence thereof to an organism, improvement or treatment with the above-described polygene transcription or more can be achieved. The effect of the target protein expression-related diseases includes diseases related to fat metabolism, diseases related to muscle synthesis, diseases associated with inflammatory factors, and the like, for example, inflammatory reaction, muscular dystrophy, obesity, metabolic syndrome, fatty liver, and the like.

The amino acid sequence encoded by the amino acid sequence of the present invention is SEQ ID No. 1 or the homologous amino acid sequence thereof can be plant extraction method, artificial synthesis method, recombinant organism production platform or any two or more of the above. A combination of methods is obtained.

The term "homologous amino acid sequence" refers to an amino acid sequence derived from the substitution, deletion or addition of a single or multiple amino acids in the amino acid sequence of a multi-peptide.

The term "plant extraction method" refers to the separation of the substance from a specific plant by the difference in solubility of the substance in different solvents. The technique of separation and purification is the technical field of the present invention and has a general knowledge. Generally known techniques, for example, separation of a multi-peptide of a specific size by a protein electrophoretic separation method, liquid chromatography, and separation using filters of different sizes. According to the previous research, a plurality of multi-peptides capable of adjusting blood sugar can be obtained from the extract of Cucurbitaceae, and the amino acid sequence of the present invention is encoded as SEQ ID No. 1. a peptide or a homologous peptide thereof, and the cucurbit plant includes, but is not limited to, bitter gourd, mountain bitter gourd, pumpkin, watermelon, courgette, gourd, and trichosanthin, and is confirmed by protein electrophoresis, an extract of the cucurbitaceae plant The multi-peptide that contains blood glucose-adjustable is a homologous protein, as shown in the first figure. In addition, the polypeptide of SEQ ID No. 1 or the homologous polypeptide thereof encoded by the amino acid sequence of the present invention can also be extracted from plants of the non-cucurbitaceae, for example: Zinnia eIegans ), Medicago truncatula, grapefruit, grape, Sambucus nigra, rice, Arabidopsis thaliana, or a combination of at least two of the above. It can be seen that the source of the amino acid sequence encoded by the present invention as the multi-peptide of SEQ ID No. 1 or the homologous multi-peptide thereof is not limited to the cucurbit plant.

The term "synthetic method" is a technique in which the amino acid is artificially linked in a manual manner to a multi-peptide by the well-known technique of the technical field of the present invention and having a general knowledge, wherein the synthetic method includes chemical synthesis. Method and peptide synthesizer. The artificial synthesis method generally has the following advantages: it is convenient to change the first-order structure of the multi-peptide, the addition of a special amino acid, and the modification of the end of the multi-peptide in the synthesis process. In general, the chemical synthesis method can be divided into a solid phase peptide synthesis method and a liquid phase peptide synthesis method, wherein the liquid phase synthesis method must perform an extraction operation after completion of the connection of each amino acid. However, since the multi-peptide intermediate obtained by the extraction is usually a mixture, the chromatographic purification step is still required. Therefore, the synthesis of the multi-peptide by liquid phase synthesis requires a complicated extraction and chromatographic purification step. In order to obtain a high purity product. The solid phase synthesis method is carried out in a solvent to carry out a bond reaction of a peptide on a solid polymer particle (or a polymeric support). In this method, the N-terminal amino acid of the desired peptide is first covalently bonded to the polymer particles, and then the subsequent amino acid is sequentially linked by means of specific bonding, and finally The multipeptide was synthesized. Since the polymer particles are not soluble in the solvent, it is only necessary to The polymer particles (and the desired peptides attached to the polymer particles) can be separated from the reagents and by-products by washing and filtration operations. Therefore, the solid phase peptide synthesis method not only has a better yield, but also can greatly shorten the reaction time because it does not require purification of the intermediate product, and is also advantageous in the synthesis of long-chain multi-peptides, and is now widely used. The peptide synthesis method used.

The term "recombinant organism production platform" refers to the construction of a nucleic acid for expressing a specific protein on a performance vector by biotechnology, and then transforming the recombinant expression vector into a host cell, such as Escherichia coli or yeast. A bacterium, a lactic acid bacterium or the like allows the recombinant expression vector to express the nucleic acid in the host cell, thereby obtaining the specific protein.

By "effective amount" is meant the amount of the compound or active ingredient required to produce the desired effect, expressed as a percentage by weight of the composition. As will be appreciated by those of ordinary skill in the art to which the present invention pertains, the effective amount will vary depending on the manner in which the particular effect is to be effected. Generally, the active ingredient or compound may be present in the compositions in an amount of from about 1% to about 100% by weight of the composition, preferably from about 30% to about 100%.

The term "pharmaceutical composition" is intended to include an effective amount of the desired compound or active ingredient to produce a particular effect, and at least one pharmaceutically acceptable carrier. As is well known to those of ordinary skill in the art to which the present invention pertains, the form of the pharmaceutical composition may vary depending on the manner in which the particular effect is to be effected, such as a lozenge, a powder, an injection, etc., and the carrier is also It is solid, semi-solid or liquid with the type of pharmaceutical composition. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerin, physiological saline, buffered saline, lanolin, paraffin, beeswax, dimethicone, ethanol.

The word "one" or "the" is used in the specification and patent application scope of the present invention. Unless otherwise stated, it shall mean one or more values.

In the following, the detailed description of the present invention will be further described in detail, but the examples are merely illustrative, and any vocabulary used herein does not limit the invention and claims. The scope and significance of the scope. In addition, the animal test in the following examples must be approved by the ethics committee of the Animal Care and Use Committee of the Chinese Medical University.

Example 1: Preparation of an amino acid sequence encoding the multi-peptide of SEQ ID No. 1.

According to the technical field of the invention and the general knowledge of the general knowledge, the amino acid sequence can be prepared by solid phase synthesis, or by recombinant organism production platform or by plant extraction, and encoded as SEQ ID No. 1. Multi-peptide, among which: the devices that can automatically synthesize multi-peptides on the market, such as: solid phase peptide synthesizer, liquid phase peptide synthesizer, and microwave peptide synthesizer, etc. The amino acid sequence disclosed in the present invention is encoded as the multi-peptide of SEQ ID No. 1, as shown in the second figure.

The expression vector containing the nucleic acid capable of transcribing the multi-peptide of the present invention is placed in a host cell by means of a recombinant organism production platform, and the nucleic acid molecule is expressed in the host cell. The amino acid sequence is encoded as SEQ ID. The peptide of No.1, as shown in the third figure, wherein the host cell may be Escherichia coli or yeast, and the expression vector may be selected from commercially available vectors, for example: pQStrep2, pQStrep4, pGEX-6p1. , or pQTEV, etc.

In addition, in the case of bitter gourd, the bitter gourd extract is first obtained, and the multi-peptide of the present invention is obtained by purifying the bitter gourd extract by a conventional technique such as protein electrophoresis purification or chromatographic purification step, and further, if necessary, A preservative such as sodium benzoate or salicylic acid is added and the polypeptide is stored at -80 °C. The steps to obtain the extract are as follows: First, the bitter gourd in the solvent Maceration is performed to obtain a crude suspension, which may be a phosphate buffer, a citrate buffer, or water, etc., and a stirrer or a grinder may be used to dissociate bitter gourd. Next, the particles in the crude suspension are removed from the liquid phase at a centrifugal speed of 12,000 revolutions per minute (rpm) to 15,000 rpm, and the filter material has a pore diameter of 0.1 μm to 0.5 μm. To filter the supernatant. Then, the obtained filtrate is sequentially passed through a filter of 1 kilodalton (kDa), a supernatant fraction thereof, and a filter membrane of 10 kilodaltons, and a filtrate portion thereof is taken, thereby obtaining the present invention. The water-soluble bitter gourd extract of the multi-peptide, wherein the filter may be selected from conventional filter products, such as Amicon filters and Millipore filters.

Example 2: Multi-peptide system for multi-site, multi-gene target reaction in vivo

In this example, the following materials were used:

(A) Subjects: The following animal tests have been approved by the Ethics Committee of the Animal Care and Use Committee of the China Medical University. Mice of the FVB strain were used for experiments, and these mouse strains were all provided by the National Laboratory Animal Center.

(B) Polypeptide: The amino acid sequence encoded in Example 1 was encoded as the multi-peptide of SEQ ID No. 1.

The FVB strain mice were divided into two groups: the control group and the experimental group. The mice in the experimental group each contained 2.5 mM/kg body weight (mmol/kg). The amino acid sequence of the present invention was encoded as SEQ ID. No. 1 multi-peptide solution (20 μl) for seven consecutive days; while in the control group, 20 μl of phosphate buffer per mouse was administered. After the end of the experiment, tissues such as jejunum, muscle, fat, liver, and kidney of each group of mice were taken out, and multi-organ and multi-target analysis was performed by system biology. A DNA microarray is known to those skilled in the art and is well known to those skilled in the art. The types of genes that are regulated on the whole genome can be observed, and the path of influence and action of the bioinformatics software can be further utilized. Therefore, the mechanism of microarray and gene expression profiling will be further explored to observe the present invention. The uncovered amino acid sequence encodes the gene affected by the multi-peptide of SEQ ID No. 1. The operation flow is described in detail as follows:

(I) RNA samples Cellular total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Next, total RNA was quantified using a Beckman DU800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA), and samples with an A260/A280 ratio greater than 1.8 were further evaluated using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The quality of its total RNA. Only when the RNA integrity number of the sample is higher than 8.0 will it be used for the microarray experimental analysis described below.

(B) Experimental analysis of DNA microarrays. DNA microarray experimental analysis methods are described in the report of Cheng et al. (2007). Briefly, total RNA (5μg) using the first MessageAmp ^TM aRNA kit (Ambion), be amplified by in vitro transcription (in vitro transcription) step. The amplified RNA (amplified RNA, abbreviated as aRNA) is then chemically reacted with the Cy5 dye, and the Cy5 stain is calibrated to the aRNA, so that the aRNA becomes the target with the fluorescent cursor. After calibration is complete fluorescent, reaction buffer using a hybrid (hybridization buffer) and coverslips Union provided by the company, the subject fluorescent calibration was reacted with hybrid Whole Genome OneArray ^TM. After a night of heterozygous reaction at 50 ° C, the non-specifically bound target was removed from the wafer by three cleaning steps. The wafers were then dried by centrifugation and scanned for fluorescence intensity using an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA, USA). We used the genepix 4.1 software (Molecular Devices) to analyze the Cy5 fluorescence intensity at each point on the wafer. The signal at each point is corrected for its intensity by subtracting the surrounding background value. We remove the probe as an intrinsic control or a point where the signal-to-noise ratio is less than zero. The points through these thresholds are normalized by the R program's limma package (Smyth, 2005).

(3) Using biological information software to analyze the role of the machine and the impact path. Analyze differentially expressed genes using bioinformatics software such as Medical Subject Headings (MeSH) (http://www.nlm.nih.gov/mesh/meshhome.html) and BibIioSphere Pathway Edition software The role of the machine, the path of influence and the degree of connection with the disease.

The analysis results obtained by the respective routes are shown in the following Table 1. From the results of Table 1, it can be seen that the amino acid sequence encoded by the amino acid sequence of the present invention as SEQ ID No. 1 can indeed be in different parts of the individual and different gene targets. The point reacts and affects the message path and disease.

Example 3: Polypeptides are used to treat conditions that are inflammatory or contain an inflammatory response

In the present example, the luminescence image measurement in the living body of the animal, the luminescence image measurement of the animal in vitro, and the immunohistochemical staining analysis are respectively confirmed, wherein the following materials are used:

(A) Subject: The luciferase (luc) gene-transferred mouse regulated by nuclear factor-κB (NF-κB) was mated with wild-type mouse F1 to produce NF-κB/luc with FVB genetic traits. The heterozygous gene was transferred to mice for experimentation.

(B) Polypeptide: The amino acid sequence encoded in Example 1 was encoded as the multi-peptide of SEQ ID No. 1.

(1) Determination of cold light images in animals in vivo

A total of 15 female transgenic mice aged 6-8 weeks were randomly divided into three groups. The first group was a blank group; the second group was a control group; the third group was an experimental group. Each of the mice in the second group and the third group was administered with a solution of lipopolysaccharide (LPS) of 4 mg/kg body weight (100 μl) by intraperitoneal injection, and the first group was administered. 100 μl of phosphate buffer in each of the mice. After 5 minutes, each of the mice in the third group was given 0.5 mg/kg body weight of the multi-peptide solution, 20 μl, and each of the mice in the first group and the second group was administered 20 μm. Lit water. Four hours later, the luminescence activity of each group of mice was observed by living images.

In detail, each of the groups is given by intraperitoneal injection. The mouse contains 150 mg/kg body weight of luciferin. After 5 minutes, each group of mice was anesthetized with isoflurane, and each group of mice was placed face up in the camera compartment of the IVIS Imaging System 200 series (IVIS Imaging System_ 200 Series Xenogen, Hopkinton, MA). The camera is set to the highest sensitivity for imaging for 1 minute. The number of photons emitted by the tissue was quantified by Living Images software (Xenogen, Hopkinton, MA), and the number of photons emitted by the gene-transferred mouse was quantified. The results are shown in Figure A, where the signal intensity The calculation is the total number of photons measured by the tissue, and the result of the signal strength is presented in photons per second (photons/sec). The cold light values of each group of mice were compared and analyzed, and the results are shown in Figure 4B.

It is known from the previous literature that NF-κB/luc heterozygous gene transgenic mice can indeed activate their nuclear factor kappa B after induction by lipopolysaccharide, and the nuclear factor kappa B transcription factor and activated nuclear factor kappa B signaling pathway are linked to It plays an important role in regulating the immune response. When nuclear factor kappa B is activated, it stimulates the gene expression of inflammatory response-related factors. As can be seen from the results of the fourth panel A and B, the average luminescence value of the first group is 2.92 × 10 ⁷ photons / sec; the intensity of the luminescence signal of the second group is stronger than that of the first group, and the average luminescence value is 31.97. ×10 ⁷ photons/sec; and the luminescence intensity of the third group was significantly lower than that of the second group, and the average luminescence value was 19.82 × 10 ⁷ photons/sec. Therefore, as a result of the above-mentioned living body, it is known that the lipopolysaccharide can induce an inflammatory reaction in the transgenic mouse, and the abdomen has a significant luminescence activity, and the amino acid sequence encoded by the present invention is encoded as SEQ ID No. 1. After treatment with the peptide, the luminescence activity of the abdomen of the transgenic mouse is decreased. The lipopolysaccharide can increase the luminescence value of the gene-transferred mouse by 10.95 times, and the multi-peptide of the present invention can achieve a cold light value of about 38% inhibition ratio.

Accordingly, the multi-peptide system encoded by the amino acid sequence of the present invention as SEQ ID No. 1 can effectively reduce the activity of nuclear factor kappa B, thereby effectively inhibiting the acute systemicity caused by external stimulation in the living body. The effect of an inflammatory response.

(II) Determination of cold light images of animals in vitro

Each group of mice in Example 3 (1) was first administered intraperitoneally to each of 150 mg/kg body weight of luciferin. After 5 minutes, each group of mice was sacrificed and quickly The tissue is removed and contains brain, heart, lung, liver, spleen, stomach, kidney, ovary and intestine. The living images were set up in the camera compartment of the IVIS Imaging System 200 series (IVIS Imaging System_200 Series, Xenogen, Hopkinton, MA) as described in Example 3 (1). Imaging, and using the Living Images software (Xenogen, Hopkinton, MA) to quantify the number of photons emitted by the tissue, the results are shown in Figure 5, where the signal intensity is calculated as the total number of photons measured by the tissue. The result of the signal strength is presented in photons per second (photons/sec). According to the results of the fifth figure, the intensity of cold light in each organ of the group of mice is different. Among them, except for the ovary, the cold light intensity measured by the control mice in the other organs is significantly higher. The blank group of mice came to increase. Compared with the control group, the mice in the experimental group were found to have a significant decrease in the intensity of luminescence in the organs of the lung, liver, kidney, and intestine.

To further understand the main organ of the multi-peptide to reduce the inflammatory response, each will The luminescence values of the organs in this group are presented in Fold change, as shown in Table 2 and Table 3 below, wherein the fold change in Table 2 = the average cold light value of the second group, the average cold light value of the first group, and the multiple of Table 3. Change = the third group of average luminescent values. The second group of average luminescent values.

From the above results, it can be seen that lipopolysaccharide can induce inflammation of organs in the transgenic mouse, and has obvious luminescence activity, but the amino acid sequence disclosed in the present invention is encoded as SEQ ID No. More than 1 peptide can reduce the luminescence activity of organs such as lung, liver, kidney, intestine, etc., indicating that the amino acid sequence encoded by the present invention encoded as SEQ ID No. 1 can indeed reduce lipopolysaccharide The activity of the induced nuclear factor kappa B is effective in inhibiting inflammation of the organs.

(C) immunohistochemical staining analysis

Organs taken from each group of mice are paraffin-embedded according to the technical field of the present invention and known to those skilled in the art, and the paraffin-embedded organs are cut into 5-μm sections and dewaxed with xylene. After dehydration with gradient alcohol, and using 3% hydrogen peroxide solution It took 15 minutes to eliminate the activity of endogenous peroxidase, and then acted at 1% bovine serum albumin for 1 hour at room temperature to block the non-specific link and then dilute 50 times. Mouse monoclonal antibodies against p56, TNF-α or IL-1β were treated at 4 ° C for 16-18 hours, and the next day, biotinylated secondary antibodies (Zymed Laboratories, South San Francisco, CA) were added. ) was allowed to stand at room temperature for 20 minutes. Finally, each of the sections was placed in an avidin-biotin complex reagent according to the procedure of the manual (Histostain®-Plus Kit, Zymed Laboratories, South San Francisco, CA) at 3, 3'- The aminobenzidine was dyed and the results are shown in the sixth graph.

As can be seen from the results of the sixth panel, compared with the tissue sections of the blank group of mice, the lipopolysaccharide-induced control group had many brown cells which reacted with TNF-α and IL-1β in the tissue sections, and The brown area is significantly increased. However, in the tissue sections of the experimental group, the brown area of the reaction with TNF-α and IL-1β was significantly less than that of the control group. It has been previously disclosed in the literature that TNF-α and IL-1β are pro-inflammatory inflammatory hormones and cause an inflammatory response. Therefore, the above results show that the amino acid sequence of the present invention is encoded as SEQ ID No. The peptide system effectively inhibits the inflammation induced by lipopolysaccharide by inhibiting the production of pro-inflammatory cytokines.

Example 4: Multi-peptide is used to inhibit liver fat deposition

In this example, the following materials were used:

(A) Subjects: Experiments were carried out using mice of the FVB strain, all of which were provided by the National Laboratory Animal Center.

(B) Polypeptide: The amino acid sequence encoded in Example 1 was encoded as the multi-peptide of SEQ ID No. 1.

Thirty FVB strains were randomly divided into three groups. The first group was a blank group; the second group was a control group, fed with high-fat diet; the third group was an experimental group with high feeding. Fat feed; and, weekly, by intraperitoneal injection, each mouse containing 10 mM/kg body weight (mmol/kg) of the amino acid sequence of the present invention encoded as SEQ ID No. 1 The peptide solution (100 microliters) was administered twice for four weeks, while each of the mice in the first and second groups was administered 100 microliters of phosphate buffer.

After the end of the experiment, after the liver tissues of each group of mice were taken out, according to the general knowledge of the technical field of the present invention and the general knowledge, the liver tissues of each group of mice were stained with H&E and oil red (oil red). O) Dyeing was observed for the accumulation of fat in the inside, and the results are shown in Figures 7 and B, respectively. And, according to the procedure of immunohistochemical staining described in the above third embodiment, the liver tissues of each group of mice were respectively treated with 4-hydroxynonenal (HNE) antibody and malonaldehyde (MDA) antibody. Immunohistochemical staining was performed to detect lipid peroxidation products in liver cells of each group of mice, and the results are shown in Figures 8 and B.

As can be seen from the results of the seventh panel, compared with the mice of the first group, the liver tissues of the mice of the second group showed significant fat vacuoles and a large amount of fat accumulation. The liver tissue of the mice in the third group had less fat accumulation than the mice in the second group.

As can be seen from the results of the eighth graph, the liver tissues of the second group of mice have more lipid peroxidation products such as 4-hydroxydecenoic acid and malondialdehyde than the first group of mice, compared with the second group. The lipid peroxidation products in the liver tissues of the mice in the third group were significantly reduced.

It can be known from the prior literature that when there are too many intracellular fat peroxidation products, cell damage such as cytopathic and fibrosis is caused, and 4-hydroxydecenoic acid and malondialdehyde are lipid peroxidation products, and Detection index of cellular oxidative damage. Yes, from the above results It is known that the polypeptide system encoded by the amino acid sequence encoded by the present invention as SEQ ID No. 1 can effectively improve the accumulation of fat in liver tissue and reduce the content of lipid peroxidation products in liver cells. In other words, the multi-peptide system encoded by the amino acid sequence of the present invention as SEQ ID No. 1 can be used to treat or ameliorate a condition caused by fatty liver or liver damage.

Example 5: Multi-peptide is used to inhibit the accumulation of fat

(A) In the present example, the following materials were used: Experimental subjects: mice that used a gene defect to cause a large accumulation of abdominal fat, which were provided by the National Laboratory Animal Center. .

(B) Polypeptide: The peptide obtained in Example 1 and having the amino acid sequence shown in Table 1 was used.

The mice were divided into two groups: the control group and the experimental group. Each week, the mice in the experimental group were given an amount of 2.5 nanomoles/kg body weight (2.5 nmol/kg) of the amine of the present invention by intraperitoneal injection. The base acid sequence was encoded as the multi-peptide solution of SEQ ID No. 1 (100 microliters) twice for four weeks; in the blank group, 100 microliters of phosphate buffer per mouse was administered.

During the course of the experiment, the body weight and feed intake of each group of mice were measured periodically. After the end of the experiment, the appearance of each group of mice was observed, and the adipose tissue of each group of mice was taken out for weighing, and the fat cells were determined by section staining. The number and size are shown in the ninth to eleventh figures. The body fat ratio of each group of mice was further calculated, and the calculation formula was: (fat weight and weight) × 100%, and the results are shown in Table 4 below.

From the results of Table 4 and Figure IX to Figure 11, it can be seen that although there was no significant difference in body weight between the control group and the experimental group before and after the experiment, the fat weight of the mice in the experimental group Fat was significantly lower than that of the control mice. From this, it can be seen that the multi-peptide system encoded by the amino acid sequence of the present invention as SEQ ID No. 1 can indeed affect the biosynthesis of fat, thereby achieving the effect of preventing or treating metabolic syndrome.

Example 6: Multi-peptide is used to prevent muscle atrophy

The following materials were used in this example:

(A) Subjects: Experiments were carried out using mice lacking a large amount of abdominal fat using gene defects, and these mouse strains were all provided by the National Laboratory Animal Center.

(B) Polypeptide: The multi-peptide encoded by the amino acid sequence prepared in Example 1 was encoded as SEQ ID No. 1.

After removing the muscle tissue of each group of mice in Example 5, the muscle tissue of each group of mice was observed by H&E staining according to the general knowledge of the technical field of the present invention and the size and size of the muscle cells. The result is shown in the twelfth figure.

From the results of the twelfth graph, it was found that the control mouse group had obvious symptoms of muscle atrophy, and the muscle atrophy of the experimental group administered with the multi-peptide of the present invention was significantly changed. good. It can be seen that the multi-peptide system encoded by the amino acid sequence of the present invention as SEQ ID No. 1 can indeed affect the synthesis of muscles and achieve the effect of preventing or treating the symptoms associated with muscular dystrophy.

Example 7: Multi-peptide is used to reduce the incidence of diabetes and complications

The following materials were used in this example:

(A) Subject: Experiments were carried out using mice of non-obese diabetic mice (NOD mice), all of which were provided by the National Laboratory Animal Center.

(B) Polypeptide: The amino acid sequence encoded in Example 1 was encoded as the multi-peptide of SEQ ID No. 1.

Twenty-three mice were divided into 4 groups and fed for 20 weeks under the following conditions: The first group was a control group, a total of six, and each mouse was given 20 μl of phosphate buffer per day; the second group a total of six, each of which is orally administered to each mouse containing 0.01 micromol/kg body weight (0.01 μmol/kg) of the amino acid sequence of the present invention encoded as the multi-peptide solution of SEQ ID No. 1. (20 microliters); the third group, a total of six, is administered orally to each mouse containing 0.1 micromol / kg body weight (0.1 μmol / kg) of the amino acid sequence of the present invention encoded as The multi-peptide solution of SEQ ID No. 1 (20 μl) was once; the fourth group, a total of five, was administered orally to each mouse containing 1 μmol/kg body weight (1 μmol/kg) per day. The amino acid sequence of the invention is encoded as a multi-peptide solution of SEQ ID No. 1 (20 microliters) once.

During the course of the experiment, the survival rate, diabetes induction rate and eye lesions of each group of mice were measured periodically, and the results are shown in Table 5. After the end of the experiment, blood samples of each group of mice were taken, and blood of each group of mice was subjected to serum biochemical examination according to the general knowledge of the technical field and the general knowledge of the present invention, and the results are shown in Table 6.

From the results of Table 5, the survival rate of the first group of mice was 4/6 (66.67%), and the survival rates of the mice of the second group, the third group and the fourth group were 6/6 (100%). , 6/6 (100%), 5/5 (100%). The diabetes induction rate of the first group of mice was 2/6 (33.33%), and the diabetes induction rates of the second, third and fourth groups were 0/6 (0%) and 0/6, respectively. 0%), 1/5 (20%). In addition, the incidence of diabetic complications was 3/6 (50%) in the first group, and 0/6 (0%) in the second, third and fourth groups, respectively. 1/6 (16.66%), 1/5 (20%). From this result, it was revealed that the administration of the multi-peptide system of the present invention can effectively improve the survival rate of the individual and reduce the occurrence of diabetes and/or its complications such as eye lesions or kidney lesions.

From the results of Table 6, the blood urea nitrogen (BUN) in the first group of mice is 40.50±4.54mg/dL, the blood urea nitrogen values of the second group, the third group and the fourth group were 37.38±2.77, 38.50±4.69 and 35.88±6.33mg/dL, respectively. The serum creatinine (Creatinine) of the first group of mice was 0.70±0.05 mg/dL, and the serum creatinine values of the mice of the second group, the third group and the fourth group were 0.64±0.12, 0.69±0.06, and 0.69±, respectively. 0.06. From the results, it was revealed that the administration of the multi-peptide of the present invention can significantly reduce the serum creatinine value and the urea nitrogen content, and as the dosage is increased, the decrease is more pronounced.

From the results of Tables 5 and 6, it can be seen that the multi-peptide system encoded by SEQ ID No. 1 by the amino acid sequence of the present invention can improve the survival rate, the diabetes induction rate, and the diabetic complications. As well as kidney damage and the like, the amino acid sequence encoded by the present invention as the SEQ ID No. 1 can be used as a pharmaceutical composition for treating or preventing diabetes or/and its complications.

The above is only the detailed description of the present invention by the examples, and any simple modifications or changes made to the embodiments of the specification should be made without departing from the spirit of the invention. The resulting hunter.

<110> China Medical University

<120> Use of multi-peptide for the production of a multi-effect pharmaceutical composition

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 68

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 1

Claims (7)

A use of a multi-peptide for the manufacture of a pharmaceutical composition for treating or/and preventing a disease associated with a target, wherein the multi-peptide comprises a sequence encoded as SEQ ID No. 1, and the target system A group consisting of a proliferative factor activating receptor, a nuclear factor kappa B, and a Sirt1 gene is selected. The use according to the first aspect of the invention, wherein the multi-peptide is an amino acid sequence encoding the multi-peptide of SEQ ID No. 1. The use according to the first aspect of the patent application, wherein the disease is muscular dystrophy. The use according to claim 1, wherein the disease is a condition having an inflammatory response. The use according to the first aspect of the patent application, wherein the disease is associated with metabolic syndrome. The use according to claim 5, wherein the disease is fatty liver. The use according to claim 5, wherein the disease is obesity.