KR20220154642A - Novel use of composition of Klotho in tacrolimus-induced renal cellular toxicity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting tacrolimus-induced renal toxicity, or preventing or treating mitochondrial diseases containing a composition comprising Klotho as an active ingredient. The Klotho compound shows a protection effect with respect to mitochondria damage in renal cells induced by tacrolimus which is an immunosuppressant, thereby improving renal functions. Accordingly, the Klotho compound can be used as a transplant disease medicine as an anti-aging functional agent and is effective in mitochondria-related diseases.

Description

Novel use of composition of Klotho in tacrolimus-induced renal cellular toxicity}

The present invention relates to a pharmaceutical composition for inhibiting tacrolimus-induced renal toxicity or for preventing or treating mitochondrial abnormalities of a composition containing Klotho as an active ingredient.

Tacrolimus, a recently developed calcineurin inhibitor, has been reported to be more effective than cyclosporine in reducing the survival rate of transplanted kidneys and reducing acute rejection within one year.

Tacrolimus, which is used as an immunosuppressant in kidney transplant patients, is a drug with a very narrow treatment area and severe individual differences. Tacrolimus is a drug that serves as a substrate for P-glycoprotein (P-gp), which is responsible for drug absorption in vivo, and Cytochrome P4503A, which is responsible for metabolism.

Despite the increase in the growth rate of short-term survival after kidney transplantation, about 3-5% of transplanted kidneys are lost every year due to the nephrotoxicity of long-term transplanted kidneys and the death of recipients. Recently, acute rejection has decreased, but adverse effects related to long-term immunosuppressive therapy are increasing. Therefore, reducing the toxic reaction of immunosuppressive agents has become the main goal of transplant recipient treatment.

Immunosuppressants that block signal transduction pathways in T cells are most commonly used as immunosuppressive drugs currently used. However, there are problems in that side effects such as renal dysfunction occur.

Therefore, there is a need to develop new therapeutic agents for improving the immune response that have no side effects and have excellent therapeutic effects.

Korean Patent Publication No. 10-2016-0014887

Therefore, in order to solve the above problems, an object of the present invention is to provide a pharmaceutical composition for suppressing renal toxicity by an immunosuppressive agent, comprising Klotho peptide consisting of amino acids of SEQ ID NO: 1 as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating abnormal mitochondrial diseases, comprising the Klotho peptide consisting of amino acids of SEQ ID NO: 1 as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for suppressing kidney toxicity by an immunosuppressive agent, comprising the Klotho peptide consisting of amino acids of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the Klotho peptide may be encoded by the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the immunosuppressive agent may be tacrolimus.

The present invention provides a pharmaceutical composition for preventing or treating abnormal mitochondrial diseases, comprising the Klotho peptide consisting of amino acids of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the mitochondrial abnormalities include Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, Friedreich's ataxia, epilepsy, peripheral neuropathy, optic neuropathy, autonomic neuropathy, Nerve-derived bowel dysfunction, sensorineural deafness, nerve-derived bladder dysfunction, migraine, ataxia, renal tubular acidosis, dilated cardiomyopathy, steatohepatitis, hepatic failure, lactic acidosis, mitochondrial encephalomyopathy encephalopathy with lactic acidemia and strokelike episodes (MELAS), Leber's hereditary optic neuropathy (LHON), MERRF syndrome, Mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), NARP (neuropathy, ataxia, and retinitis pigmantosa), Barth syndrome Syndrome), Leigh Syndrome and Kans-Syres Syndrome.

The present invention relates to a novel use of a compound containing a Klotho component, wherein the Klotho compound is effective in improving renal function by showing a protective effect against mitochondrial damage in renal cells induced by the immunosuppressive agent Tacrolimus , As an antioxidant, it is effective not only for the treatment of transplant diseases but also for mitochondria-related diseases.

1 is a result of measuring the viability of cells exposed to tacrolimus in order to confirm the protective effect of Klotho against cell damage of cells exposed to tacrolimus.
2 is a result of measuring the total amount of active oxygen to confirm the effect of Klotho on the increase in active oxygen in cells exposed to tacrolimus.
Figure 3 is the result of analyzing the effect of Klotho on the mitochondrial membrane potential difference (ΔΨm) reduction by tacrolimus.
Figure 4 is the result of analyzing the effect of Klotho on the reduction of intracellular oxygen consumption (OCR) by tacrolimus.

Definitions of terms used in the present invention are as follows.

“Subject” or “patient” means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects participating in clinical research trials who do not show any clinical signs of disease or subjects participating in epidemiological studies or subjects used as controls.

“Immunosuppressive agent” or “immunosuppressive compound” refers to a compound capable of depleting the population size of T and/or B clones of lymphocytes or inhibiting their reactivity, expansion or differentiation. Immunosuppressive compounds include, but are not limited to: Cyclosporine (also known as "CsA", marketed as Neoral or Sandimmune) and tacrolimus (also known as "FK506", marketed as Prograf), including purine metabolism inhibitors such as mycophenolate mofetil (also known as "MMF", marketed as Cellcept) and azathioprine (marketed as Azasan or Imuran); everolimus (marketed as Certican); ) and proliferation inhibitors such as sirolimus (also known as “rapamycin” or “Rapa”, marketed as Rapamune®) monoclonal antibodies (“mAb”) such as anti-CD45 and anti-CD45RB (ref: U.S. Pat. No. 7,160,987) "); Monoclonal antibodies against T-cells, such as OKT3 Monoclonal antibodies against IL-2 receptor, including humanized anti-TaT antibodies, such as basilixamab and daclizumab T-cells, such as CTLA-4-Ig1 fusion protein substances that block costimulatory pathways, such as substances that can induce chimerism (i.e., the coexistence of donor and recipient immune cells that recognize the graft tissue as self) and cyclophosphamide (marketed as Cytoxan) Non-myeloblative pre-transplant therapy. For a discussion of immunosuppressive agents and their targets see Stepkowski (2000) Expert Rev. Mol. Med. June 21, 2000: 1-23. In the present invention, in particular, tacrolimus (also known as "FK506", marketed as Prograf) is used as an immunosuppressive agent.

"Inhibitor or inhibitor" means a substance that inhibits, blocks or reduces a particular activity. The mechanism of action of the inhibitor is not particularly limited. Examples may include organic or inorganic compounds, polymeric compounds such as proteins, carbohydrates and lipids, and composites of various compounds.

"Adjuvant composition for treating and preventing immune diseases" refers to a composition that helps to induce a desired immune response by promoting the activity of various known immunosuppressants used for the treatment and prevention of immune diseases or reducing side effects. However, the mechanism of action is not particularly limited. Examples may include organic or inorganic compounds, polymeric compounds such as proteins, carbohydrates and lipids, and composites of various compounds.

The term "synergy" means that the effect that occurs when each component is administered in combination (combination) is greater than the sum of the effects that occur when each component is administered alone, or that a beneficial additional effect is greater. including performing

The term "administered in combination" or "administered in combination" means that the compounds or components are administered together to the subject animal. Administration of each compound or component together means that each component can be administered at the same time or in any order or sequentially at different times to obtain the desired therapeutic effect.

An "effective amount" is an appropriate amount that produces beneficial or desirable clinical or biochemical results. An effective amount can be administered once or more. For purposes of this invention, an effective amount is an amount suitable for temporarily alleviating, ameliorating, stabilizing, reversing, slowing or delaying the progression of a disease state. A composition is said to be "pharmaceutically or physiologically acceptable" if the recipient animal is able to tolerate administration of the composition, or if administration of the composition to the animal is suitable. An agent can be said to be administered in a "therapeutically effective amount" when the amount administered is physiologically important. An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

"Functional food" means a food whose functionality is improved by adding the composition of the present invention to common food. Functionality can be roughly divided into physical properties and physiological functions. When the composition of the present invention is added to general foods, the physical properties and physiological functions of general foods will be improved, and the present invention comprehensively treats foods with such improved functions as 'functional foods'. ' is defined as

A "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue.

A “diluent” is defined as a compound that is diluted in water which will dissolve the compound as well as stabilize the biologically active form of the subject compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state improvement or palliation and relief (partial or total), detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or prophylactic methods. The treatments include treatment required for disorders that have already occurred as well as disorders that are prevented. "Palliating" a disease means reducing the extent and/or undesirable clinical signs of the disease state and/or slowing or prolonging the time course of the disease compared to no treatment. means to lose Therefore, the reduction of cytotoxicity by the tacrolimus in mammals is

(1) reducing oxidative stress;

(2) reducing apoptosis;

(3) reduction of toxicity in renal epithelial cells and beta cells;

(4) improving pancreatic function;

(5) reduction of renal cell toxicity and tissue fibrosis, and

(6) Recovery of kidney function

Includes one or more of the efficacies composed of.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Hereinafter, the present invention will be described in detail.

In the present invention, the present inventors first identified the use of Klotho peptides for suppressing renal toxicity by an immunosuppressive agent.

In particular, the Klotho compound can reduce cytotoxic reactions and unwanted side effects caused by long-term administration of an immunosuppressive agent such as tacrolimus, as well as provide an effective method for improving renal function.

<Tacrolimus and Klotho>

The present invention relates to the function of Klotho compounds involved in the immunosuppressive response of immunosuppressants. In particular, the immunosuppressive agent is most preferably tacrolimus.

Tacrolimus (known as FK506, marketed as Prograf) is an immunosuppressive agent that binds to FKBP-12, an intracellular protein, and inhibits cellular immunity (activation of T lymphocytes). It is a macrolide antibiotic found in the fermentation broth of Streptomyces tsukubaensis, a microorganism obtained from a soil sample, and has an immunosuppressive effect. It is structurally different from the currently most widely used immunosuppressive agent, CsA (cyclosporin Publication Patent Publication 10-2016-0014887A), but its mechanism of action is known to be similar, that is, it suppresses the gene expression of the effecting cell to show its effect and Tacrolimus first binds to a type of immunophilin called FK506-binding protein (FKBP) and inhibits the activity of calcineurin, a phosphatase (also called phosphatase 2B).

<Tacrolimus chemical formula>

Currently, the tacrolimus is primarily used to prevent organ rejection in patients receiving allogeneic liver, kidney or heart transplants. Tacrolimus prolongs host and graft survival in animal transplant models of liver, kidney, heart, bone marrow, small intestine and pancreas, lung and trachea, skin, cornea and extremities. In animals, tacrolimus suppresses some humoral immunity and greatly inhibits cell-mediated responses such as allograft rejection, delayed-type hypersensitivity, collagen-induced arthritis, experimental allergic encephalomyelitis, and graft-versus-host disease.

However, in the treatment of immune diseases using the immunosuppressant tacrolimus, side effects such as toxic reactions of the immunosuppressant and increased frequency of diabetes tend to appear, which is problematic.

The present invention relates to the function of improving side effects on the immune response caused by an immunosuppressive agent such as tacrolimus of a composition containing Klotho peptide as an active ingredient. In particular, it is useful for improving cytotoxicity problems showing oxidative stress and increased apoptosis among side effects on the immune response.

The Klotho peptide may be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention, or may be purchased and used commercially.

Unless otherwise specified below, the term Klotho peptide according to the present invention is used as a concept including all of the compound itself, pharmaceutically acceptable salts, hydrates, solvates, isomers and prodrugs thereof.

"Pharmaceutically acceptable salt" means a formulation of a compound that does not cause significant irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The terms "hydrate", "solvate" and "isomer" also have the same meaning. The pharmaceutical salt is the compound of the present invention, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, inorganic acids such as phosphoric acid, sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, triclo It can be obtained by reacting with an organic carboxylic acid such as rhoacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like. In addition, by reacting the compound of the present invention with a base, salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, N-methyl-D-glue It can also be obtained by forming salts of organic bases such as carmine and tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine.

A "hydrate" is a compound of the present invention that contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces; means its salt.

"Solvate" means a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents in this regard are solvents that are volatile, non-toxic, and/or suitable for administration to humans.

"Isomer" means a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but differs optically or sterically.

"Prodrug" means a substance that is transformed in vivo into the parent drug. Prodrugs are often used because, in some cases, they are easier to administer than the parent drug. For example, they may obtain bioactivity by oral administration whereas the parent drug may not. A prodrug may also have improved solubility in a pharmaceutical composition than the parent drug. For example, prodrugs are esters that facilitate passage through cell membranes ("prodrugs"), which are hydrolyzed by metabolism to the active carboxylic acid in cells where water solubility is detrimental to mobility, but once water solubility is beneficial. It will be a compound administered as a "drug"). Another example of a prodrug would be a short peptide (polyamino acid) attached to an acid group that is metabolized to reveal an active site.

Hereinafter, the present invention will be described in more detail by examples. The following examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation example>

As a reagent used in the present invention, Tacrolimus (TAC) is used by dissolving Prograf (Astellas Pharma, Ibaraki, Japan) sold by Astellas Pharmaceuticals in DMSO (Sigma-Aldrich, St. Louis, MO, USA), and recombinant human Klotho ( R&D Systems, Minneapolis, MN, USA) reagent was diluted in PBS.

<Example 1>

Human kidney-2 cell line culture

The human kidney-2 cell line used in the present invention was purchased from the American Type Culture Collection (ATCC, Manassas, VA, UAS). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Wisent, St. Bruno, Que., Canada) containing 10% fetal bovine serum (FBS; Wisent), 100 U/mL penicillin, and 100 mg/mL streptomycin (Wisent). were cultured in a 37°C incubator in the presence of 5% CO2.

<Example 2>

Cell viability measurement

For cell viability experiments, 2 x 10 ⁴ /0.5mL of cells were dispensed into a 96 well plate (SPL Life Sciences, Pocheon, Korea), and the next day, TAC was added in DMEM medium without FBS at 40, 80 μg/mL and Klotho was treated at a concentration of 1 μg/mL and then incubated for 12 and 24 hours. As a control, equal amounts of DMSO and PBS were treated under the same conditions. Cell survival in each condition was measured by adding 10 μl of cell counting kit-8 reagent (Dojindo Molecular Technologies, Rockville, MD, USA) to each well, reacting in a 37°C incubator for 2 hours, and then ELISA Microplate Reader (Molecular Devices , Sunnyvale, CA, USA) was used to measure absorbance at a wavelength of 450 nm. Cell viability was expressed as percentage (%) compared to control.

<Example 3>

Measurement of Klotho concentration in culture medium

In order to examine the difference in Klotho concentration secreted by cells, the drug was treated and reacted, and the culture medium was collected and carried out according to the method recommended by the kit using a sandwich ELISA kit (R&D Systems) that can measure human Klotho. . Absorbance was measured at a wavelength of 450 nm using an ELISA Microplate Reader (Molecular Devices).

<Example 4>

Reactive oxygen species (ROS) measurement

For the measurement of reactive oxygen species (ROS), HK-2 cells were dispensed at 1 x 10 ⁶ /mL in a 12 well plate, treated with the reagent under the same conditions as the viability measurement, reacted, and washed 1-2 times with PBS. After reaction with 10 μM H2DCFDA (Molecular probes, Carlsbad, CA, USA) or 0.5 μM Mitosox Red (Molecular probes) for 1 hour in a 37°C incubator, the cells were harvested with 0.05% Trypsin and 0.53 mM EDTA (Wisent) and Dissolve in mL of PBS and read using FACSCalibur (BD Bioscience, San Jose, CA, USA) in the channels of FL-1H (Excitation 488 nm and Emission 513-535) and FL-2H (Excitation 550 nm and Emission 574), respectively. analyzed.

<Example 5>

Apoptosis measurement

After processing and reacting with the drug, wash 1-2 times with PBS, harvest the cells with 0.05% Trypsin and 0.53 mM EDTA (Wisent), float in binding buffer (BD Bioscience), centrifuge, and FITC-Annexin 5 μl of -V culture reagent (BD Bioscience) was diluted in 100 μl binding buffer and reacted at room temperature for 15 minutes. 400 μl of binding buffer was added to each sample, and read and analyzed using FACSCalibur (BD Bioscience).

<Example 6>

Mitochondrial membrane potential difference (ΔΨm) measurement

After treating and reacting with the drug, washing with PBS 1-2 times, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1; Cayman Chemical., Ann Arbor, MI, USA) into 5 μl/mL media and reacted in a 37°C constant temperature incubator for 20 minutes. The excitation/emission wavelengths of JC-1 monomer and JC-1 aggregates were read and analyzed using FACSCalibur (BD Bioscience) at 490/530 nm and 525/590 nm, respectively.

<Example 7>

Measurement of cellular oxygen consumption

2 × 10 ⁴ cells were seeded in an XF24 cell culture microplate (Seahorse Bioscience, North Billerica, MA, USA), and the next day, before measuring the oxygen consumption rate (OCR) of the cells, the medium was replaced with bicarbonate-free medium. , and cultured for 1 hour in a Non-CO2 cell incubator. Four respiratory chain inhibitors, including 1 μM oligomycin, 0.5 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A in the XF cell Mito Stress Test kit (Seahorse Bioscience) were sequentially treated, and again OCR change amount was measured. Measurement results were analyzed using Wave for XF ^e Instrument Controllers (Version 2.2.0276, Seahorse Bioscience) by reflecting the cell protein concentration of each well.

statistical treatment

The experimental results in the present invention were expressed as mean ± standard deviation, and in statistical analysis using ANOVA one-way analysis, significance was placed at P < 0.05.

<Experimental Example 1>

Protective effect of Klotho against cell damage caused by Tacrolimus (TAC)

As a result of analyzing the viability of cells exposed to tacrolimus, in cells treated with tacrolimus, the cell viability decreased over time and showed a tendency to decrease as the exposure concentration increased (see Fig. 1A). It was confirmed that the concentration was significantly decreased in cells exposed to TAC 60 μg/mL (TAC 60) compared to the control group (DMSO) (see FIG. 1B). In addition, it was confirmed that in the group in which Klotho was added at a concentration of 1 to 2 μg/mL to TAC 60 μg/mL, the cell viability was significantly recovered compared to the control group (see FIG. 1C), and cell death by apoptosis was investigated with Annexin V. Results It was confirmed that the Annexin V-positive cells increased in the TAC group were significantly decreased in the cell group exposed to incorporation of Klotho (see Fig. 1D) (*P<0.05vs.DMSO,#P<0.05vs.TAC40)

<Experimental Example 2>

Effect of Klotho on Tacrolimus (TAC)-induced Reactive Oxygen (ROS) Increase

It was confirmed that the increase in the total amount of reactive oxygen species (total ROS) and superoxide anion, which is a reactive oxygen species (ROS) generated in mitochondria, by tacrolimus was significantly reduced in the cell group exposed to incorporation of Klotho (see FIG. 2). (*P<0.05 vs. DMSO.)

<Experimental Example 3>

Effect of Klotho on reduction of mitochondrial membrane potential difference (ΔΨm) by Tacrolimus (TAC)

Mitochondrial membrane potential difference (ΔΨm) was confirmed as the ratio of the number of JC-1 Red fluorescence-positive cells. As a result, the number of JC-1 Red fluorescence-positive cells in cells exposed to tacrolimus significantly decreased compared to the control group, DMSO, while Klotho was incorporated. In the case of one group, it was confirmed that a significantly high value was measured (see FIG. 3). (*P<0.05 vs. DMSO.)

<Experimental Example 4>

Effects of Klotho on Reduction of Intracellular Oxygen Consumption (OCR) by Tacrolimus (TAC)

In order to investigate the effect of Klotho on mitochondrial function, OCR was measured directly in the cell culture medium in which live cells were cultured. It was confirmed that the group treated with the recovery showed the result (see FIG. 4A). Basal respiration, ATP synthesis (ATP linked), and maximal respiration were calculated based on the A graph in which oligomycin, FCCP, and antimycin A/rotenone were sequentially treated, and the combined group of TAC and Klotho showed TAC It can be seen that these values are significantly improved compared to those alone, indicating that Klotho has an effect of restoring the mitochondrial function reduced by TAC (see Fig. 4B). (*P<0.05 vs. DMSO.)

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (4)

A composition for inhibiting toxicity of renal cells caused by tacrolimus, comprising Klotho peptide, which is composed of the amino acids of SEQ ID NO: 1 and encoded by the nucleotide sequence of SEQ ID NO: 2, as an active ingredient. According to claim 1,
The composition for inhibiting toxicity of renal cells caused by tacrolimus, wherein the Klotho peptide improves the function of renal cells through a protective effect against mitochondrial damage in renal cells induced by tacrolimus. A composition for inhibiting mitochondrial damage in renal cells caused by tacrolimus, comprising the amino acid of SEQ ID NO: 1 and the Klotho peptide encoded by the nucleotide sequence of SEQ ID NO: 2 as an active ingredient. According to claim 3,
The Klotho peptide is a composition for inhibiting mitochondrial damage in renal cells caused by tacrolimus, which restores mitochondrial function deteriorated by tacrolimus and reduces increased oxidative stress.

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