Therapeutic agent for immune cell migration-caused disease and method for screening same
11378572 · 2022-07-05
Assignee
Inventors
- Sunghoon Kim (Seoul, KR)
- Nam Hoon Kwon (Gyeonggi-do, KR)
- Jin Young Lee (Seoul, KR)
- Kun Ho Kim (Gyeonggi-do, KR)
Cpc classification
A61K31/713
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
International classification
G01N33/50
PHYSICS
Abstract
The present invention relates to a therapeutic agent for immune cell migration-caused disease and a method for screening the same and, more particularly, to a pharmaceutical composition comprising a KRS inhibitor (or expression or activity inhibitor) as an effective ingredient for preventing or treating an immune cell migration-related disease, a method for controlling the migration of immune cells by regulating a level of KRS in immune cells, a cell membrane site-specific moiety level of KRS or the migration of KRS to the cell membrane, and a method for screening a therapeutic agent for immune cell migration-caused disease, using KRS. According to the present invention, the migration of immune cells can be controlled by means of KRS, which can find very useful applications in the prevention, alleviation, and treatment of immune cell migration-related disease.
Claims
1. A method for identifying an agent for treating an immune cell migration-related disease selected from the group consisting of a cardiovascular disease, a fibrotic disease, an inflammatory disease, and Alport disease, the method comprising: contacting an immune cell selected from the group consisting of a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil, a dendritic cell, a natural killer cell, a megakaryocyte, a T cell, and a B cell with a laminin and a candidate agent selected from the group consisting of a siRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acid (PNA), antisense nucleotide, antibody, aptamer, peptide, peptide mimetic, substrate analog, natural extract, and synthetic compound, wherein the immune cell is contacted with the laminin and the candidate agent simultaneously, or the immune cell is contacted sequentially with the candidate agent followed by the laminin or with the laminin followed by the candidate agent; performing an assay to measure a level of lysyl tRNA synthetase (KRS) at the plasma membrane of the immune cell or a level of KRS translocated to the plasma membrane of the immune cell, wherein the KRS comprises an amino acid sequence as set forth in SEQ ID NO: 1; and identifying an agent that lowers the level of KRS at the plasma membrane of the immune cell or the level of KRS translocated to the plasma membrane of the immune cell relative to the immune cell prior to the contacting step, whereby an agent for treating the immune cell migration-related disease is identified.
2. The method of claim 1, wherein the assay comprises separating the cytosol and membrane fraction of the immune cell after the contacting step and measuring a first level of KRS in the separated cytosol and a second level of KRS in the membrane fraction.
3. The method of claim 2, wherein measuring the first and second levels of the KRS comprises binding an anti-KRS antibody to KRS present in the separated cytosol and membrane fraction.
4. The method of claim 3, comprising transferring the separated cytosol and membrane fraction to a solid support and incubating the anti-KRS antibody with the solid support under conditions sufficient to bind the anti-KRS antibody to KRS present on the solid support.
5. A method for identifying an agent that reduces lysyl tRNA synthetase (KRS) at the plasma membrane of an immune cell, the method comprising: contacting an immune cell selected from the group consisting of a monocyte, a macrophage, a neutrophil, an eosinophil, a basophil, a dendritic cell, a natural killer cell, a megakaryocyte, a T cell, and a B cell with laminin and a candidate agent selected from the group consisting of a siRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acid (PNA), antisense nucleotide, antibody, aptamer, peptide, peptide mimetic, substrate analog, natural extract, and synthetic compound, wherein the immune cell is contacted with the laminin and the candidate agent simultaneously, or the immune cell is contacted sequentially with the candidate agent followed by the laminin or with the laminin followed by the candidate agent; performing an assay to measure a level of lysyl tRNA synthetase (KRS) at the plasma membrane of the immune cell or a level of KRS translocated to the plasma membrane of the immune cell, wherein the KRS comprises an amino acid sequence as set forth in SEQ ID NO: 1; and identifying an agent that reduces KRS at the plasma membrane of the immune cell or KRS translocated to the plasma membrane of the immune cell relative to the immune cell prior to the contacting step, whereby an agent that reduces lysyl tRNA synthetase (KRS) at the plasma membrane of the immune cell or KRS translocated to the plasma membrane of the immune cell is identified.
Description
BRIEF DESCRIPTION OF DRAWINGS/FIGURES
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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MODE FOR CARRYING OUT INVENTION
(53) Hereinafter, the present invention will be described in more detail with reference to examples, experimental examples and manufacturing examples. However, the following examples, experimental examples and preparation examples are illustrative of the present invention, and the present invention is not limited to the following examples, experimental examples and manufacturing examples.
Example 1: The Role of Laminin Signaling in the Immune Cell Migration and Infiltration
(54) Among several extracellular matrix (ECM), it was examined which ECM promotes the migration and infiltration of immune cells, typically monocytes/macrophages. A transwell migration assay was performed using collagen (Col), fibronectin (FN) and laminin (LN) as extracellular matrices, and detailed methods were as follows. Transwells (Corning, #3421-5 mm) were coated with gelatin (0.5 mg/ml) and RAW 264.7 cells (1×10.sup.5 cells/well) were seeded into the top chambers. Serum free DMEM (500 μl) containing 10 μg/ml of laminin (laminin mixture, Biolamina), fibronectin or collagen, respectively, was placed in the bottom chambers. After 24 hours, cells were fixed with 70% methanol for 30 minutes and stained with 50% hematoxylin for 30 minutes. After removing non-migrating cells from the top of the membrane with a cotton swab, the membrane was taken and mounted on the slide. Migrating cells on the underside of the membrane were observed and quantified under a high magnification microscope.
(55) As shown in
Example 2: Effect of Laminin Subtypes on the Immune Cell Migration and Infiltration
(56) Effect of laminin subtypes on the immune cell migration and infiltration was evaluated. A transwell migration assay was performed in the same manner as in Example 1 using LN111, LN211, LN221, LN411, LN421, LN511, and LN521 as various laminin subtype proteins (purchased from Biolamina). Specific sequences of laminin subtypes are referred to α4 chain of SEQ ID NO:4, α2 chain of SEQ ID NO: 10, α5 chain of SEQ ID NO: 11, β2 chain of SEQ ID NO:6, β1 chain of SEQ ID NO: 12, γ1 chain of SEQ ID NO: 8. according to the chain forming each laminin subtype,
(57) As shown in
Example 3: Translocation of KRS from the Cytosol to the Plasma Membrane Induced by Treatment of Laminin in Immune Cells
(58) After dispensing RAW 264.7 cells (2×10.sup.6 cells) in 100 mm dish and incubating for 18 hours, cells were treated with LN421 l g/ml in serum free DMEM media and harvested at 0 hour, 12 hour, 24 hour. Proteins of RAW 264.7 cells were separated into the cytosol and membrane fractions using ProteoExtract Subcellular Proteome Extraction Kit (Calbiotech, cat #539790). Obtained proteins were electrophoresed and transferred to PVDF membrane (Milipore) and blocked with 3% skim milk. KRS was then detected by western blot. Specifically, KRS polyclonal antibody (rabbit, Neomics, Co. Ltd. #NMS-01-0005) was added and reacted for 1 hour. Unbound antibody was removed and the membrane was added and reacted with anti-rabbit secondary antibody (ThermoFisher Scientific, #31460). After reacting with the secondary antibody, films were exposed in the dark room using ECL reagent as a substrate. Photosensitized bands were compared to the standard molecular markers to identify the bands corresponding to the size of KRS. Antibodies against Na+/K+ ATPase (Abcam, ab76020) and tubulin (Santa cruz SC-5286) were used to identify the plasma membrane and cytosol markers, respectively.
(59) As shown in
Example 4: Effect of KRS on the LN421-Dependent Immune Cell Migration and Infiltration
(60) To determine whether KRS influences the LN421-specific immune cell (especially monocyte/macrophage) migration, macrophages transformed to enhance or suppress KRS expression were treated with LN421, respectively, and a transwell migration assay was performed. As a control, leucyl-tRNA synthetase (LRS, SEQ ID NO:3), a protein similar to KRS, was used.
(61) Specifically, KRS- or LRS-overexpressing macrophages were prepared as follows: KRS (SEQ ID NO:1)-Myc, LRS (SEQ ID NO:3)-Myc inserted in pcDNA3, respectively, were transfected into Raw 264.7 cells using Turbofect (ThermoFisher Scientific) (48 hours). Cells transfected with Ev (empty vector, pcDNA3)-Myc were prepared as a negative control.
(62) Macrophages with suppressed KRS or LRS expression were prepared as follows: si-KRS (SEQ ID NO: 13) and si-LRS (SEQ ID NO:20) were transfected into Raw 264.7 cells, respectively, using Lipofectamin (ThermoFisher Scientific) (72 hours). As a negative control, cells transfected with si-control (si-RNA duplex with medium GC content (Invitrogen, Cat No. 12935-300)) were prepared.
(63) Thus prepared transformed cells were examined and verified for upregulation or downregulation of KRS or LRS expression using western blot for each protein (data not shown).
(64) For each of the transformed macrophages, a transwell migration assay was performed in the same manner as in Example 1 using 1 μg/ml of laminin 421.
(65) As shown in
Example 5: Screening of Compounds Inhibiting the Immune Cell Migration: Compounds Inhibiting the Translocation of KRS to the Plasma Membrane
(66) Based on the results of Example 3 and Example 4, it was understood that not only the expression level of KRS but also the intracellular behavior of KRS significantly influences the LN421-dependent migration of monocyte/macrophage. In particular, the phenomenon in which KRS translocates to the plasma membrane, and increase its level in the membrane-specific manner in immune cells was considered to be an important pathology for the immune cell migration and infiltration-related diseases. Therefore, the aim of this study was to verify that inhibition of such pathological behavior of KRS could be one of the therapeutic strategies for immune cell migration and infiltration-related diseases. On the other hand, KRS is an organ necessary for synthesizing proteins in cells under normal conditions. Therefore, simply increasing or decreasing the amount of KRS is likely to be inadequate as a practical treatment strategy due to concerns about side effects on normal functioning. Thus, the present inventors screened compounds that affect intracellular kinetics, expression and activity in various aspects of KRS, and examined whether they can specifically inhibit the migration of monocyte/macrophage without side effects.
(67) In particular, the screening method provided herein was used to find compounds that inhibit the translocation of KRS to the plasma membrane, and to identify and examine their therapeutic effects on diseases related to the immune cell migration. The specific methods are as follows.
(68) First, in order to determine whether various KRS inhibitor candidates could exert inhibitory effect on the KRS translocation to the plasma membrane, RAW 264.7 cells (2×10.sup.6 cells) were dispensed in 100 mm dishes and incubated for 18 hours, followed by treatment with laminin 421 1 μg/ml in serum free DMEM, and 100 nM of each of various KRS inhibitor candidates, and cells were further incubated for 12 hours. After harvesting, proteins of RAW 264.7 cell were separated into the cytosol and membrane fractions using ProteoExtract Subcellular Proteome Extraction Kit (Calbiotech, cat #539790). Obtained proteins were electrophoresed and transferred to PVDF membrane (Milipore) and blocked with 3% skim milk. Afterwards, KRS was detected by western blot, and the specific method were as described in Example 3.
(69) It was possible to determine tentatively that the inhibitor candidate actually suppressed the KRS translocation when the level of KRS was reduced specifically in the membrane fraction as relative to the cytosol fraction after the inhibitor treatment, by comparing the amount of KRS in each of the cytosol and membrane fractions before and after the treatment with inhibitor candidates.
(70) Thus identified agents as inhibiting the translocation of KRS to the plasma membrane was added to the LN421-treated macrophages to perform a transwell migration assay. Through this, it was examined whether inhibition of KRS translocation to the plasma membrane had any inhibitory effect on the LN421-specific monocyte/macrophage migration. Specifically, transwells (Corning, #3421-5 mm) were coated with gelatin (0.5 mg/ml) and RAW 264.7 cells (1×10.sup.5 cells/well) were seeded in the top chambers. 500 μl of serum free DMEM containing 1 μg/ml of laminin 421 (LN421, Biolamina) was placed in the bottom chambers. Thereafter, DMSO, or KRS inhibitor compounds (in DMSO) were treated at various concentrations (30 nM, 100 nM, 300 nM, 1 μM, 3 μM, respectively) in the upper chambers. After 24 hours, cells were fixed with 70% methanol for 30 minutes and stained with 50% hematoxylin for 30 minutes. After removing non-migrating cells from the top of the membrane with a cotton swab, the membrane was taken and mounted on the slide. Migrating cells on the underside of the membrane were observed and quantified under a high magnification microscope.
(71)
(72) In addition, as shown in
(73) In the following in vivo experiments regarding immune cell migration-related diseases, BC-KI-00053 compound was used as a representative inhibitor candidate.
Example 6: Effect of the Inhibitor of KRS Translocation to the Plasma Membrane on the Monocyte/Macrophage Infiltration in the In Vivo Acute Inflammatory Responses
Example 6-1: Ear Skin Wound Model
(74) To investigate the effect of the inhibitor of KRS translocation to the plasma membranes during monocyte infiltration in acute inflammatory responses, an ear skin wound model using CX3CR1-GFP mouse (Stock no. #005582, Jackson Laboratory, Bar Harbor, USA) was prepared. Monocytes, macrophages and Langerhans cells appear green in CX3R1-GFP mice. Mice were administered orally with vehicle or BC-KI-00053 (100 mg/kg, dissolved in vehicle, once daily) for a total of 4 days from 2 days prior to imaging (D−2, D−1, D−0, D+1). For a vehicle, corn oil:polyethylene glycol 400:Tween 80:methyl cellulose (1%)=20:30:1:49 was used. A 31G syringe was used to puncture the skin of the ear (time D−0) to induce acute inflammatory responses. Blood vessels were labeled using anti-CD31 antibody bound to Alexa Flour 555 (identifiable as red). Confocal microscopy was used as an imaging equipment.
(75) As shown in
Example 6-2: Liver Ischemia-Reperfusion Injury Model
(76) A liver ischemia-reperfusion injury model was prepared using CX3CR1-GFP mice to investigate the effect of inhibitors of KRS translocation to the plasma membrane on the monocyte infiltration during ischemic immune responses. Monocytes, macrophages and Kupffer's cells appear green in CX3CR1-GFP mice. Mice were orally administered with vehicle or BC-KI-00053 (100 mg/kg, dissolved in vehicle, once daily) for a total of three days beginning two days before imaging (D-2, D-1, D-0). For vehicle, corn oil:polyethylene glycol 400: Tween 80: methyl cellulose (1%)=20:30:1:49 was used. On day 3 of oral administration (D-0), triad (bile duct, hepatic artery, hepatic vein) occlusion was performed using a 6-0 suture as shown in
(77) As shown in
Example 7: Therapeutic Effect of the Compound Inhibiting KRS Translocation to the Plasma Membrane in In Vivo Liver Fibrosis
(78) Hepatocytes appear red in Actin-DsRed mice (Stock no. #006051, Jackson Laboratory (Bar Harbor, USA)). In order to induce liver fibrosis in this mouse, CCl.sub.4 (carbon tetrachloride) was dissolved in corn oil and injected intraperitoneally twice a week at a concentration of 20% for a total of 6 weeks. Three weeks after start of CCl.sub.4 administration, vehicle and BC-KI-00053 (100 mg/kg) were administered orally, daily for three weeks. For vehicle, corn oil:polyethylene glycol 400:Tween 80:methyl cellulose (1%)=20:30:1:49 was used. Animal groups were set up as shown in Table 1 below.
(79) The degree of fibrosis on the surface and inside (area with a depth of 30-50 μm) of liver was detected by the Second Harmonic Generation (SHG) technique of intravial imaging (Excitation: 780 nm, Detection: 390 nm).
(80) TABLE-US-00001 TABLE 1 Number of Group Treatment animals Control (1) Corn oil-treated animal (normal) + 1 vehicle administration (2) Corn oil-treated animal (normal) + 1 BC-KI-00053 administration Experiment (3) CC1.sub.4 liver fibrosis animal + 2 vehicle administration (4) CC1.sub.4 liver fibrosis animal + 3 BC-KI-00053 administration
(81) In the normal animal group administered with BC-KI-00053 (animal group (2) in Table 1), no weight loss was seen and no other symptoms occurred in the liver. Therefore, BC-KI-00053 compound was considered to be innocuous in vivo. Only the fibrosis animals administered with vehicle (animal group (3) in Table 1) died early 4 weeks after start of the experiment due to toxicity of CCl.sub.4. Specifically, as shown in
(82) As shown in
Example 8: Therapeutic Effect of an Inhibitor of KRS Translocation to the Plasma Membrane in In Vivo Liver Fibrosis
(83) Experimental Methods
(84) 1) Preparation of a Pulmonary Arterial Hypertension (PAH) Model and Administration of Test Compounds
(85) In order to induce PAH in 6-week-old female SD rats (Oriental Bio), 60 mg/kg of MCT (monocrotaline), a substance that causes pulmonary hypertension through pulmonary arterial injury, was injected subcutaneously. Then animals were divided into four groups (5 animals per group), and orally administered with vehicle, sildenafil (25 mg/kg, once daily) or BC-KI-00053 (25 or 50 mg/kg, dissolved in vehicle, once daily) for 3 weeks. Vehicle was corn oil:polyethylene glycol 400:Tween 80:methyl cellulose (1%)=20:30:1:49.
(86) 2) Measurements of Blood Flow and Pressure
(87) Three weeks later, rats were anesthetized with isoflurane, and blood flow and pressure were measured using an MPVS cardiovascular pressure and volume system (model name: MPVS Ultra, manufactured by Millar Instruments). Right ventricular systolic pressure (RVESP) and diastolic pressure, left ventricular systolic pressure and diastolic pressure were measured using a dedicated catheter (Mikro-Tip rat pressure catheter, manufactured by Millar Instruments). Cardiac output was measured using a perivascular blood flow probe (Transonic® Flowprobes, manufactured by Millar Instruments), and experimental techniques were performed in the same manner as described in the literature: Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass D A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc 2008, 3(9):1422-34.
(88) 3) Immunohistochemistry (IHC)
(89) IHC staining for CD68, a monocyte/macrophage marker, was performed using lung tissues from each experimental group. Collected lungs were fixed in PFA (paraformaldehyde) according to the conventional procedure, and then embedded in paraffin through water washing, dehydration, and tissue clearing processes. Lung tissue paraffin blocks of rats were cut to a thickness of 6 m and slides were prepared. Thereafter, staining was performed as follows. First, three times for 5 minutes xylene treatment, 2 minutes in 100% ethanol twice, 95% ethanol, 90% ethanol, 70% ethanol, DW treatment for 2 minutes in this order and washed with PBS for 5 minutes (2 times). After treatment with 0.3% H.sub.2O.sub.2 (10 minutes), slides were washed twice with PBS for 5 minutes. Then slides were soaked in 0.01M citrate buffer of pH 6.0 and microwaved for 3 minutes and 30 seconds, then antigen retrieval of cooling for 10 seconds and reheating for 10 seconds was repeated for 10 minutes followed by 20 minutes of cooling at room temperature. Afterwards, slides were washed three times for 5 minutes with PBS-T (0.03% Triton-X). After 30 minutes blocking (2% BSA & 2% goat serum in PBS) at 4° C. anti-CD68 antibody (1:200, Abcam, ab31630) was treated overnight at 4° C. After washing three times with PBS-T for 5 minutes, slides were treated with polymer-HRP anti-mouse envision kit (DAKO) for 1 hour at 4° C. After washing 3 times with PBS-T for 5 minutes, 1 ml of DAB substrate buffer and 20 ul of DAB chromogen were mixed and treated with tissue. After 10 minutes when colors developed, slides were washed twice with tertiary distilled water. Stained tissues were treated with Mayer's hematoxylin (Sigma) for 1 minute, and then treated in the order of 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol, twice for 2 minutes for each solution. Finally, after three times of xylene treatment for 5 minutes, cover slides were mounted using a mounting solution, and observed with an optical microscope.
(90) Results
(91) Pulmonary hypertension causes right ventricular pressure to rise due to narrowing of the pulmonary artery, resulting in right ventricular failure. In addition, if the reward mechanism is destroyed by persistent hypertension, right ventricular hypertrophy occurs followed by right ventricular enlargement. This results in compression of the left ventricle due to displacement of the ventricular septum and reduction of the left ventricular dilatation volume and cardiac output (Lee Woo-seok et al. Clinical Characteristics and Prognostic Factors in Patients with Severe Pulmonary Hypertension. Korean Circulation J 2007, 37:265-270). Ultimately, pulmonary hypertension is primarily associated with the right ventricle but also with the function of the left ventricle.
(92) As shown in
(93) In addition, there was no decrease in the left ventricular end systolic pressure (LVESP) following the treatment of BC-KI-00053, but rather, LVESP was increased in the BC-KI-00053 50 mg/kg administration group as shown in
(94) Taken together, administration of the inhibitor of KRS translocation to the plasma membranes (particularly BC-KI-00053) provided by the present invention not only exhibits therapeutic and alleviating effects on PAH, but also poses a relatively low risk of developing side effects of existing therapeutic drugs.
(95) In addition, as shown in
Example 9: Therapeutic Effect of the Inhibitor of KRS Translocation to the Plasma Membrane on In Vivo Hypertension-Induced Proteinuria, Glomerulosclerosis, Kidney and Heart Fibrosis
Example 9-1: Effect of the Inhibitor of KRS Translocation to the Plasma Membrane on Hypertension Kidney Damage, Heart Damage and Fibrosis Development in the FHH Rat of Superimposed Hypertension
(96) Experimental Methods
(97) Experiments were performed using male FHH rats of 9-12 weeks of age. These animals were provided by the University of Mississippi Medical Center and approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Rats were fed ad libitum, and provided with a purified AIN-76 rodent feed containing 0.4% NaCl (Dyets, Bethlehem, Pa.) after weaning. Fawn-hooded hypertensive (FHH) rat is a genetic model of spontaneous hypertension associated with glomerular hyperfiltration and proteinuria. In order to promote glomerular damage in this rat, DOCA strips were implanted after single (one) kidney extraction.
(98) Specifically, FHH rats were anesthetized with isoflurane and telemetry transmitters (model TA11PAC40, Data Sciences International, St. Paul, Minn.) were implanted as described in ‘Williams, J. M. et al. Im J Physiol Regul Integr Comp Physiol (2012)’. Briefly, surgery was performed under 2% to 3% isoflurane-O.sub.2, and the catheter of the device was inserted into the left femoral artery and guided upstream to the aorta. Body part of the telemetry unit was placed in the lateral cavity of the left leg and sutured with muscle tissue. Skin was then closed. To prevent infection, animals were given Baytril (10 mg/kg) and Rimadyl (5 mg/kg), a long-acting analgesic to control surgical pain. After surgery, rats were housed in individual cages in a quiet air-conditioned room environment with a 12:12 hour light-dark cycle and it took a week to fully recover from surgery. Thereafter, the basic mean arterial blood pressure (MAP) and proteinuria were measured for 4 hours (10 am to 2 μm) before the rats were housed in the metabolic cage. Proteinuria was measured using the Bradford method and BSA (Bio-Rad Laboratories, Hercules, Calif.) as a standard.
(99) One week after the transmitter insertion, rats were uninephrectomized as described in Wang, X. et al. Am J Physiol Renal Physiol (2016). Briefly, rats were anesthetized with 2-3% isoflurane-O.sub.2 and the right flank was dissected in aseptic condition. The right kidney was gently lifted and threaded tightly around the renal vessels and ureters. The right kidney was extracted by cutting the distal ends of the renal vessels and ureters. The incision was closed with a continuous subcutaneous stitch, after which the skin was further closed. After the rat's right kidney was removed, DOCA pellets (200 mg, Innovative Research of America) was subcutaneously implanted in the neck.
(100) After single kidney extraction and DOCA transplant surgery, rats had recovery time for 3 days. Rats were provided with water containing 1% NaCl in place of distilled water, and randomly divided into two groups: Group 1 (n=15) was administered with BC-KI-00053 (25 mg/kg daily) by gastrointestinal gavage; Group 2 (n=15) was administered with the same volume (2.5 ml/kg daily) of vehicle (corn oil, polyethylene glycol 400, Tween 80 and methylcellulose) by gastrointestinal gavage. Blood pressure and proteinuria were measured weekly for 3 weeks in the experimental group. At the end of the experiment, rats were anesthetized with isoflurane and blood samples were taken to measure creatinine levels. Rats were then flushed with 50 ml of 0.9% NaCl through aorta and perfused with 20 ml of 4% paraformaldehyde. Kidneys and hearts were collected for histological evaluation.
(101) Paraffin sections prepared with a thickness of 3 m were stained with Masson's trichrome to measure the degree of glomerular damage and renal interstitial fibrosis. Images were obtained using a Nikon Eclipse 55i microscope and NIS-Elements D 3.0 software equipped with Nikon DS-Fil color camera (Nikon, Melville, N.Y.). The degree of glomerular damage was assessed by the blinded experimenter, rating from 0 to 4+ for 30-40 μlomeruli/section. 0 represents normal glomeruli, 1+ represents 1˜25% loss, 2+ represents 26˜50% loss, 3+ represents 51˜75% loss, and 4+ indicates more than 75% loss of capillaries in the tufts. Cortical and medulla fibrosis were analyzed using NIS-Elements automated measurement software after thresholding to determine the percentage of images stained in blue. In addition, immunohistochemical staining (IHC) for CD68, a monocyte/macrophage marker for kidney tissue, was performed in the same manner as in Example 8.
(102) Statistics: Each data is expressed as mean±SEM. Comparisons between groups were analyzed by two-tailed test. P value p<0.05 was considered statistically significant.
(103) Results
(104) There was no difference in basal body weight between the vehicle treatment group and BC-KI-00053 treatment group (control group 309.57±4.14 g, experimental group 304.7±5.39 g, p>0.05). Body weight was reduced by approximately 10% in vehicle or BC-KI-00053-treated rats during the study period, but there was no statistical difference between the two groups (
(105) MAP data measured via telemetry in control and experimental FHH rats are shown in
(106) Data for proteinuria in FHH rats of the control and experimental groups are shown in
(107) Data of plasma creatinine concentrations in FHH rats of the control and experimental are shown in
(108) Uninephrectomy with DOCA pellet insertion followed by switching to 1% NaCl water in FHH rats had a morphologically significant effect on glomeruli and coronary injury (
(109) As seen in the sectioned tissue samples stained with Sirius red (
(110) In addition, as shown in
Example 9-2: Effect of the Inhibitor of KRS Translocation to the Plasma Membrane on Hypertension Kidney Damage, Heart Damage and Fibrosis Development in the Dahl SS (Salt Sensitive) Rat
(111) Experimental Methods
(112) Experiments were performed using male Dahl SS rats at 9-12 weeks of age. These animals were provided by the University of Mississippi Medical Center and approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Rats were fed ad libitum, and these rats were provided with a purified AIN-76 rodent feed containing 0.4% NaCl (Dyets, Bethlehem, Pa.) after weaning. Dahl salt-sensitive (SS) rat is an animal model that rapidly develops high hypertension, proteinuria, glomerulosclerosis and renal interstitial fibrosis on high salt (HS) diet.
(113) Dahl SS rats were anesthetized with isoflurane and telemetry transmitters (model TA11PAC40, Data Sciences International, St. Paul, Minn.) were aseptically implanted in the same manner as described above. After surgery, rats were housed in individual cages in a quiet air-conditioned room environment with a 12:12 hour light-dark cycle and it took a week to fully recover from surgery. Then, the baseline mean arterial blood pressure (MAP) was measured before the rats were placed in the metabolic cage to measure urine protein excretion. Proteinuria was measured using the Bradford method and BSA (Bio-Rad Laboratories, Hercules, Calif.) as a standard.
(114) Rats were then randomly divided into two experimental groups: Group 1 (n=15) was treated with BC-KI-00053 (25 mg/kg daily) by gastrointestinal gavage; Group 2 (n=15) was administered by gastrointestinal gavage with the same volume (2.5 ml/kg per day) of vehicle (corn oil, polyethylene glycol 400, Tween 80 and methyl cellulose). Simultaneously with the administration of agents, feed was changed to HS feed containing 8% NaCl (Dyets, Bethlehem, Pa.) and blood pressure and proteinuria were measured at 7, 14 and 21 days after starting HS feed. At the end of the experiment, rats were anesthetized with isoflurane and blood samples were taken to measure creatinine levels. Rats were then flushed with 50 ml of 0.9% NaCl through the aorta and perfused with 20 ml of 4% paraformaldehyde. Kidneys and hearts were collected for histological evaluation.
(115) Paraffin section preparation and evaluations of the degree of glomerular damage, cortex and medulla fibrosis were performed as described above. In addition, immunohistochemical staining (IHC) for CD68, a monocyte/macrophage marker for kidney tissue, was performed in the same manner as in Example 8.
(116) Statistics: Each data is expressed as mean±SEM. Comparison between groups was analyzed by two-tailed test. P value p<0.05 was considered statistically significant.
(117) Results
(118) There was no difference in baseline body weight between the vehicle- and BC-KI-00053-treated groups (control group 337.92±9.86 g, experimental group 350.13±9.173 g, p>0.05). Body weights were maintained or increased slightly in vehicle- or BC-KI-00053-treated rats, but there was no statistical difference between two groups during the entire study period (
(119) MAP data measured via telemetry in the control and experimental Dahl SS rats are shown in
(120) Data of proteinuria in the control and experimental Dahl SS rats are shown in
(121) Data of plasma creatinine concentrations in the control and experimental Dahl SS rats are shown in
(122) Providing an HS diet had a significant effect on the glomerular and coronary injury morphologically in Dahl SS rats (
(123) As seen in the sectioned samples stained with Sirius red (
(124) In addition, as shown in
Example 10: Effect of the Inhibitor of KRS Translocation to the Plasma Membrane on Kidney Fibrosis and Immune Cell Infiltration in the Animal Model of In Vivo Alport Syndrome
(125) The experiment was conducted using 129Sv/J mice (Boys town hospital). Animals groups were (i) 129Sv/J wild-type mice with vehicle administration (0.5% methyl cellulose suspension), (ii) 129Sv/J Alport mice (COL4A3 knockout mouse, Cosgrove D et al., Genes Dev. 1996 Dec. 1, 10(23):2981-92) with vehicle administration (0.5% methyl cellulose suspension) (iii) 129Sv/J Alport mice with BC-KI-00053 administration. Each animal group consists of two mice. BC-KI-00053 was dissolved in 0.5% methyl cellulose suspension and orally administered at a concentration of 100 mg/kg, and kidney fibrosis and the immune cell infiltration were evaluated. Each animal group was treated with a control substance or a test agent once a day from 3 weeks of age for a total of 4 weeks. After 4 weeks of treatment, kidney paraffin sections were stained with collagen I (a marker of fibrosis) and CD45 to observe the extent of leukocyte infiltration. Evaluation of fibrosis and infiltration was performed in the same manner as in the above examples.
(126) As can be seen in
Example 11: Effect of Controlling the Immune Cell Migration/Infiltration by Anti-KRS Antibody
(127) It was examined whether an antibody specifically binding to KRS has an effect of controlling immune cell migration/infiltration. In this experiment, an antibody consisting of a heavy chain of SEQ ID NO:21 and a light chain of SEQ ID NO:23 was used representatively as an anti-KRS antibody. In the present specification, the antibody was referred to as N3 (monoclonal) antibody.
(128) The specific experimental methods are as follows. Transwell (Corning #3421-5 mm) was coated with gelatin (0.5 mg/ml), and then RAW 264.7 cells (1×10.sup.5 cells/well) were seeded into the top chambers. Serum free DMEM (500 μl) containing laminin 421 (1 μg/ml) was placed in the bottom chambers. Anti-KRS antibody (N3 antibody) was treated at 100 nM concentration in the top chambers. After 24 hours, cells were fixed with 70% Methanol for 30 minutes and then stained with 50% hematoxylin for 30 minutes. After removing non-migrating cells in the upper part of the membrane with a cotton swab, the membrane was taken and mounted on the slide. Migrating cells present on the underside of the membrane were observed under a high magnification microscope (
(129) In addition, RAW 264.7 cells were treated with laminin 421 (1 μg/ml) and anti-KRS antibody (100 nM) for 24 hours and harvested. Then samples were prepared by separating into the cytosol and membrane fractions using ProteoExtract subcellular proteom extraction kit (Calbiochem), and subjected to western blot. Specific methods of western blot is as described in Example 3.
(130) As a result, it was confirmed that anti-KRS antibody (N3 antibody) effectively inhibited the LN421-dependent monocyte/macrophage migration, which is shown in
(131) On the other hand, the present inventors found out that KRS translocated from the cytoplasm to the plasma membrane, and KRS in the plasma membrane sometimes got embedded in the membrane with a part of N-terminal regions of the protein exposed to the extracellular space (typically 1 to 72 amino acid residues in the N-terminal regions of KRS (preferably, SEQ ID NO:1)). Accordingly, it is thought that an antibody which can bind to the N-terminus of KRS among anti-KRS antibodies could have significant advantages in vivo in terms of inhibiting the immune cell migration. Of course, it is apparent to those skilled in the art that even an anti-KRS antibody targeting different regions of KRS other than the extracellularly exposed region can be used for treatment because it can still inhibit KRS activity through further treatment for its intracellular penetration.
(132) Representatively, N3 antibody is the antibody capable of binding to the N-terminus of KRS, and the treatment of this antibody specifically decreased KRS level in the plasma membrane of immune cells (
Example 12: Therapeutic Effect of Anti-KRS Antibody in the In Vivo PAH Model
(133) Experimental Methods
(134) 1) Preparation of a PAH Model and Administration of Test Compounds
(135) To induce PAH in 7-week-old SD rats (Orient bio), 60 mpk of MCT (monocrotaline) was subcutaneously injected. Then rats were divided into 4 groups (5 animals in each group), and administered with either of 1 mpk of mock human IgG (Thermo Fisher Scientific, negative control), 1 mpk or 10 mpk of anti-KRS antibody (N3 antibody), 25 mpk of sildenafil (positive control) for 3 weeks. All antibodies were intravenously injected twice a week and sildenafil was orally administered everyday.
(136) 2) Measurements of Blood Flow and Pressure
(137) Three weeks later, rats were anesthetized with isoflurane, and blood flow and pressure were measured using an MPVS cardiovascular pressure and volume system (model name: MPVS Ultra, manufactured by Millar Instruments). Right ventricular systolic pressure (RVESP) and diastolic pressure, left ventricular systolic pressure and diastolic pressure were measured using a dedicated catheter (Mikro-Tip rat pressure catheter, manufactured by Millar Instruments). Cardiac output was measured using a perivascular blood flow probe (Transonic® Flowprobes, manufactured by Millar Instruments), and experimental techniques were performed in the same manner as described in the literature: Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass D A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc 2008, 3(9):1422-34.
(138) 3) Immunohistochemistry (IHC)
(139) Collected lungs were fixed in PFA (paraformaldehyde) according to a conventional procedure, and then embedded in paraffin through water washing, dehydration, and tissue clearing processes. Lung tissue paraffin blocks of rats were cut to a thickness of 6 m and slides were prepared. Thereafter, staining was performed as follows. First, slides were treated with xylene 3 times for 5 minutes, followed by treatments with 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, DW for 2 minutes in this order and washed with PBS for 5 minutes. After treatment with 0.3% H.sub.2O.sub.2, slides were washed twice with PBS for 5 minutes. After soaking in 0.01M citrate buffer and heating, slides were washed with PBS-T (0.03% tween 20). After 30 minutes blocking at room temperature (2% BSA & 2% goat serum in PBS), tissues were stained with anti-CD68 antibody (1:200, ED1 clone, Abcam) overnight at 4° C. After washing three times with PBS-T for 5 minutes, tissues were treated with polymer-HRP anti-mouse envision kit (DAKO) for 1 hour at 4° C. After washing three times with PBS-T, color was developed by treatment with DAB substrate buffer and DAB chromogen 20. Thus stained tissues were treated with Mayer's hematoxylin (Sigma) for 1 minute, and then treated twice for 2 minutes in the order of 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol. Finally, xylene was treated three times for 5 minutes and observed with an optical microscope.
(140) Results
(141) 12-1. Changes in the Blood Pressure and Cardiac Output
(142) A PAH model, in which the immune cell infiltration is deeply related to pathology, was treated with anti-KRS antibody (N3 monoclonal antibody) at 1 mpk or 10 mpk for 3 weeks (i.e., twice a week). Subsequently, measurements of right ventricular end-systolic pressure (RVESP), right ventricular end-diastolic pressure (RVEDP), left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and cardiac output (CO) were carried out and the results are shown in Table 2.
(143) TABLE-US-00002 TABLE 2 MCT + MCT + MCT + MCT ± Mock IgG N3 Ab 1 mpk N3 Ab 10 mpk sildenafil (n = 4) (n = 5) (n = 5) (n = 5) RVESP 62.5 ± 5.7 45.0 ± 8.1 41.2 ± 7.7 48.4 ± 9.6 (mmHg) RVEDP 2.8 ± 1.5 1.4 ± 2.2 3.8 ± 1.3 2.6 ± 1.3 (mmHg) LVESP 81.5 ± 11.4 95.8 ± 4.8 93.4 ± 11.3 83.2 ± 4.7 (mmHg) LVEDP 1.0 ± 0.8 2.6 ± 1.9 4.6 ± 3.9 3.6 ± 2.3 (mmHg) CO 58 ± 4.7 74.0 ± 10.9 59.8 ± 12.9 49.6 ± 17.7 (ml/min) (n = 4) (n = 5) (n = 5) (n = 4) (1 animal in MCT + mock IgG-treated group died during anesthetization. 1 animal in the sildenafil-treated group died during surgery and CO could not be measured.)
(144) Pulmonary hypertension causes the right ventricular pressure to increase due to narrowing of the pulmonary artery, resulting in right ventricular failure. In addition, if the reward mechanism is destroyed by persistent hypertension, right ventricular enlargement is followed by right ventricular hypertrophy. This results in compression of the left ventricle due to displacement of the ventricular septum and reduction of the left ventricular dilatation volume and cardiac output (Lee Woo-seok et al. Clinical Characteristics and Prognostic Factors in Patients with Severe Pulmonary Hypertension. Korean Circulation J 2007, 37:265-270). As a result, pulmonary hypertension is primarily associated with the right ventricle but also with the function of the left ventricle.
(145) RVESP is increased in PAH patients, which was also confirmed in the PAH animal model of this experiment. In contrast, as shown in
(146) In addition, there was no decrease in left ventricular end systolic pressure (LVESP) following administration of anti-KRS antibody (N3 antibody), but rather significant increase in LVESP was observed as shown in
(147) Taken together, it was confirmed that administration of anti-KRS antibody (N3 antibody) has effects of alleviating and treating PAH symptoms, improving the possibility of side effects of existing therapeutic drugs.
(148) 12-2. Echocardiography
(149) Findings of D-shaped left ventricle suggesting pressure overload in the right ventricle were observed in three mice treated with MCT alone (i.e., non-administered PAH model) and three mice treated with MCT+sildenafil, but non in the therapeutic antibody (anti-KRS antibody)-treated group.
(150) In addition, as shown in Table 3 below, the body weight of each group was increased to a similar extent and there was no significant difference. In other words, no abnormal findings including abnormal weight loss by therapeutic antibody administration were observed.
(151) TABLE-US-00003 TABLE 3 MCT + MCT + MCT + MCT + Mock IgG Ab 1 mpk Ab 10 mpk sildenafil (n = 4) (n = 5) (n = 5) (n = 5) Absolute 101.4 ± 14.2 113.5 ± 14.6 104.1 ± 12.3 104.1 ± 26.4 change (g) Relative 48.8 ± 7.8 43.6 ± 5.2 40.7 ± 5.0 49.8 ± 10.5 change (%)
12-3. Monocyte/Macrophage Migration and Infiltration
(152) IHC staining for CD68, a monocyte/macrophage marker, was performed using lung tissues from each experimental group. As shown in
Example 13: Effect of Anti-KRS Antibody in the In Vivo Acute Lung Injury Model
(153) Experimental Methods
(154) 1) Preparation of an LPS-Induced Acute Lung Injury Model and Administration of Test Compounds
(155) The acute lung injury model was prepared by intratracheal injection of 2.5 mg/kg LPS (Sigma) into 7-week-old male C57BL/6 mice (DooYeol biotech).
(156) In order to investigate the effect of KRS inhibitors on acute lung injury, C57BL/6 mice were first intravenously injected with N3 antibody at the concentration of 1 mg/kg or 10 mg/kg, and after 24 hours, LPS 2.5 mg/kg was intratracheally injected. After 24 hours of LPS injection, each mouse was sacrificed to collect and analyze lung tissues and bronchoalveolar lavage fluid (BALF).
(157) 2) Cell Counting of Immune Cells in BALF (Bronchoalveolar Lavage Fluid)
(158) BALF obtained by washing the lungs with PBS was collected and centrifuged at 800×g for 10 minutes at 4° C. to collect pellets. After cells were suspended, red blood cells were removed using RBC lysis buffer (eBioscience cat no. 00-4333-57). After stopping the reaction with PBS, cells were washed twice, and resuspended in 400 μl PBS to measure the number of cells by hemocytometer. The number of neutrophils were counted by hema3 staining.
(159) 3) FACS of Immune Cells in the Lung Tissues
(160) Lung tissues were collected and rotated at 37° C. for 45 minutes using gentleMACS Octo Dissociator (MACS Miltenyi Biotec, order no. 130-095-937) to smash the tissues. Tissues were then filtered using a cell strainer (40 m) and centrifuged at room temperature for 5 minutes at 1500 rpm. Pellet was collected and red blood cells were removed using RBC lysis buffer (eBioscience cat. no. 00-4333-57). Cells were collected and resuspended in FACS buffer (PBS containing 1% NaN3 and 3% FBS), and 50 μl of the cell suspension was placed in a tube, mixed well with the same amount of antibody, and stained at 4° C. for 1 hour, protecting from light. FITC rat anti-CD11b (BD Pharmingen) and PE rat anti-mouse F4/80 (BD Pharmingen) antibodies were used to analyze the migration of interstitial macrophage (IM) to the lung. After washing twice at 400×g for 5 minutes using FACS buffer, it was analyzed by Navios flow cytometer (Beckman).
(161) 4) Masson's Trichrome Staining of the Lung Tissues
(162) Lung tissues were embedded in paraffin in the conventional manner and then sectioned. Thereafter, the tissue slides from which paraffin was removed using xylene was washed with DW, and then treated with Bouin fluid for 1 hour at 56-60° C. Tissues were then stained with Weigert's iron hematoxylin solution for 10 minutes, washed, and then stained again with Biebrich scarlet-acid fuchsin solution for 10-15 minutes and washed. Stained tissues were treated with phosphomolybdic-phosphotungstic acid solution for 10-15 minutes, transferred to aniline blue solution and stained for 5-10 minutes. After washing, stained tissues were treated with 1% acetic acid solution for 2-5 minutes. After washing and dehydration, stained tissues were treated with xylene and mounted.
(163) Results
(164) 13-1. Inhibitory Effect on the Immune Cell Migration in BALF
(165) As shown in
(166) In particular, as shown in
(167) 13-2. Inhibitory Effect on the Immune Cell Migration in the Lung Tissues
(168)
(169) Excessive migration and infiltration of immune cells, such as macrophages/monocytes, is an important pathology in tissue fibrotic disease. As a result of Masson's trichrome staining of the lung tissues from the acute lung injury model (
INDUSTRIAL APPLICABILITY
(170) As explained so far, the present invention relates to a therapeutic agent for immune cell migration-caused diseases and a method for screening the same and, more particularly, to a pharmaceutical composition comprising a KRS inhibitor (expression or activity inhibitor) as an effective ingredient for preventing or treating an immune cell migration-related disease, a method for controlling the migration of immune cells by regulating a level of KRS in immune cells, a level of KRS specifically present at a plasma membrane location or the translocation of KRS to the plasma membrane, and a method for screening a therapeutic agent for immune cell migration-caused diseases, using KRS. According to the present invention, the migration of immune cells can be controlled by means of KRS, which can find very useful application in the prevention, alleviation, and treatment of immune cell migration-related diseases, therefore industrial applicability is very high.