WO2023014062A1 - Hapln1을 포함하는 섬유성 질환의 예방 또는 치료용 조성물 - Google Patents
Hapln1을 포함하는 섬유성 질환의 예방 또는 치료용 조성물 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to a composition for repairing various tissue damage due to fibrosis, preventing or treating fibrotic diseases, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient. It's about Specifically, the present invention relates to a composition for repairing and regenerating damage caused by fibrosis of tissue containing HAPLN1 as an active ingredient. In addition, the present invention relates to a composition for repairing and recovering various tissue damage and functional loss due to fibrosis, and preventing or treating aging/degenerative diseases including fibrotic diseases.
- HPLN1 proteoglycan link protein 1
- the present invention discloses a composition for preventing or treating fibrotic disease and a method for preventing or inhibiting fibrosis of cells using the composition. According to the present invention, by preventing and inhibiting cell fibrosis, the occurrence or progression of various diseases caused by intervening cell fibrosis can be fundamentally suppressed as well as treated.
- 'lung fibrosis' in which the lungs harden like stones, refers to a disease in which healthy lung tissue is changed into scar tissue for some reason, causing the lung tissue to become thick or hard.
- the cause is clearly known as a specific hazardous chemical used to maintain the sterilization action in the humidifier, but the disease in which the lungs harden without knowing the cause is 'idiopathic pulmonary fibrosis (IPF)' , Idiopathic Pulmonary Fibrosis)'.
- IPF pulmonary fibrosis
- Idiopathic Pulmonary Fibrosis Idiopathic Pulmonary Fibrosis
- This disease is a kind of interstitial lung disease (ILD) that histologically shows symptoms of 'Usual Interstitial Pneumonia (UIP)' characterized by inflammation and fibrosis of the lung parenchyma, and is characterized by aging,
- ILD interstitial lung disease
- UIP 'Usual Interstitial Pneumonia
- Various risk factors are known, including smoking, gastroesophageal reflux disease, environmental/occupational exposure, dust work, radiation exposure, autoimmunity, drug addiction, hypersensitivity pneumonitis, viral/pathogen infection, and family history.
- the etiology of this disease is not clearly known.
- the average age of onset is 69 years old, and it mainly occurs in the elderly, and males are more than twice as common as females, and the prognosis is poor.
- 'idiopathic pulmonary fibrosis' a representative pulmonary fibrosis
- the number of domestic patients is 1.7 per 100,000 people, and as the cause is unknown, it is a very scary disease that is difficult to treat.
- the average survival time is 60 months, but acute exacerbation due to infection occurs in 14% of patients per year. When an acute exacerbation occurs, the survival time drops sharply to 15 months.
- the alveolar wall is thickened with scar tissue, and as the scar tissue gradually deteriorates, the lung parenchymal tissue is destroyed. Inhalation, especially inhaling air, becomes difficult at this time, making it impossible to breathe in enough air to oxygenate the blood. Eventually, the alveoli lose their original function, and rapid breathing, frequent dry cough, tiredness, and club-shaped changes appear on the fingertips and toes. Crackles are induced.
- the COVID-19 virus which has recently become popular as a pendemic, also infects and inflames the lungs, sometimes causing pulmonary fibrosis in the process of healing.
- the most well-known risk factor for fibrosis is old age.
- researchers who have been exploring lung tissue lesions have recently discovered that the lung cells of patients with pulmonary fibrosis cannot sustain division and growth, which are signs of cellular senescence.
- the cellular senescence theory believes that the aging of organisms, including humans, is due to the accumulation of senescent cells that are not physiologically useful but rather promote pathological conditions. It is a phenomenon that enters the process and no longer divides and produces cells at the normal rate.
- the effect of cellular senescence on fibrosis in elderly pneumonia is systematically well described (Yanagi 2017, Int. J. Mol. Sci. 18, 503).
- SASP serine-associated secretory phenotype
- IL-1 ⁇ interleukin 1 beta
- the present inventors paid attention to the fact that the risk factor for pulmonary fibrosis is old age, and in order to discover 'endogenous substances in blood' that can prevent or treat the onset of this disease, the disease combination between young and old mice ( While exploring through qualitative and quantitative analysis techniques using heterochronic parabiosis and proteomics technology, hyaluronan (HA) and proteoglycan link protein (HAPLN1) drew attention as the most likely substance. It became.
- HA hyaluronan
- HPLN1 proteoglycan link protein
- HAPLN1 protein is one of the constituent proteins in the extracellular matrix (ECM) first discovered in vertebrate cartilage, and plays a role in stabilizing the structural aggregate of hyaluronic acid and proteoglycan, At this time, it is reported that it acts as a hinge to make a bottle-brush shape by making the gap between the proteoglycans to be bound very densely.
- ECM extracellular matrix
- the present inventors have found that recombinant human HAPLN1 (rhHAPLN1) actually suppresses the degradation of its partner molecules, high molecular weight HA (HMW HA) and aggrecan (a type of proteoglycan), through its hinge-type linkage function, resulting in damage-associated molecules. It has been proven that it can reduce the production of low molecular weight HA (LMW HA) or 32-mer peptide as a model (DAMPs, damage-associated molecular patterns) substance.
- LMW HA low molecular weight HA
- DAMPs
- Myofibroblasts are major cells that excessively form fibrous tissue such as collagen in fibrotic diseases. These myofibroblasts characteristically show the expression of ⁇ smooth muscle actin ( ⁇ SMA), and therefore the level of ⁇ SMA expression is an important indicator for determining the level of expression of myofibroblasts.
- ⁇ SMA-expressing myofibroblasts are increasing in various fibrotic diseases such as idiopathic pulmonary fibrosis and renal fibrosis, and ⁇ SMA expression has also been reported to increase in in vivo fibrosis models. These myofibroblasts are mainly derived from fibroblasts, and are also reported to be derived from endothelial cells, epithelial cells, stem cells, etc.
- TGF ⁇ 1 is the most well-known factor for inducing differentiation of myofibroblasts (or activation of hepatic stellate cells), and in addition, mechanical stress in a stiff environment has been reported as a factor for differentiation of myofibroblasts (The American Journal of Pathology, 2007. 170(6): 1807-1816). Therefore, induction of ⁇ SMA expression by TGF ⁇ 1 in various cells is used as an in vitro fibrosis model, and the activity of reducing induced ⁇ SMA expression is judged as anti-fibrotic efficacy (Molecular Medicine 2021 27:22).
- BIPF Pulmonary Fibrosis
- pirfenidone a candidate drug, on pulmonary fibrosis
- the research team administered bleomycin three times a day for 10 days or 28 days from the day after the first administration of bleomycin (day 0) (day 1), followed by treatment.
- the efficacy of each was examined.
- the level of inflammation-related biomarkers decreased in the group treated with prednisolone, a type of anti-inflammatory corticosteroid, but the level of TGF ⁇ 1, a measure of anti-fibrotic efficacy, was not decreased.
- the present inventors have found that the HAPLN1 protein composition prevents and prevents fibrosis of various tissues even at very low concentrations using the TGF ⁇ 1-induced fibrotic disease cell model and the bleomycin-induced pulmonary fibrosis mouse model (BIPF) as described above.
- BIPF bleomycin-induced pulmonary fibrosis mouse model
- HAPLN1 for example, US Patent Publication US 2013/0052198 discloses the HAPLN1 polypeptide as one of a wide range of individual factors secreted by Marrow stromal cells (MSC), and when administered to an inflammatory disease subject, the characteristics of the disease suggests that it can weaken However, nothing has been said about the action of preventing or treating fibrosis in various tissues.
- MSC Marrow stromal cells
- the present invention relates to a composition for preventing and treating fibrotic diseases comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and preventing and treating fibrotic diseases using the same.
- HPLN1 hyaluronan and proteoglycan link protein 1
- a reagent composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it as an active ingredient it is used in the study of the disease to identify the mechanism of the disease and related disease It is intended to contribute to the development of preventive or therapeutic compositions.
- HPLN1 hyaluronan and proteoglycan link protein 1
- the present invention provides the following aspects of the invention.
- One aspect of the present invention is a pharmaceutical composition for preventing or treating fibrotic diseases comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient. it's about
- Another aspect of the present invention relates to a composition in which the protein of the composition has 80% or more sequence identity to the amino acid sequence of SEQ ID NO: 1.
- Another aspect of the present invention relates to a composition in which the nucleic acid related to the gene in the composition is included in an expression vector.
- Another aspect of the present invention relates to a composition in which the lesion of the fibrotic disease is selected from the group consisting of skin, liver, intestine, heart, lung, and kidney.
- one aspect of the present invention is that the foci of the fibrotic disease are skin fibroblasts, hepatic stellate cells, colonic fibroblasts, cardiac microvascular endothelial cells, lung fibroblasts, renal tubular cells, and renal proximal tubular epithelial cells. It relates to a composition selected from the group consisting of.
- Another aspect of the present invention relates to a composition wherein the fibrotic disease is ischemic fibrosis.
- one aspect of the present invention relates to a composition in which a single dose of the composition is 0.1 ng/ml to 500 ng/ml.
- Another aspect of the present invention relates to a composition that is administered at a dose of 0.001 to 5 mg/kg BW of rhHAPLN1 protein when the composition is administered in vivo.
- one aspect of the present invention relates to a composition that is included as a main or auxiliary active ingredient in a composition for preventing or inhibiting cellular fibrosis.
- another aspect of the present invention is to treat cells with a composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient to prevent cell fibrosis. It relates to methods of preventing or suppressing it.
- HPLN1 hyaluronan and proteoglycan link protein 1
- kits for preventing or inhibiting cell fibrosis comprising the composition according to any one of items [1] to item [9] and instructions for treatment according to the method of item [10] It is about.
- a further aspect of the present invention relates to an experimental reagent composition related to preventing or inhibiting cell fibrosis, including the composition according to any one of [1] to [9].
- composition and method of the present invention by preventing and inhibiting cell fibrosis, the occurrence or progression of various diseases caused by cell fibrosis is fundamentally inhibited, thereby increasing the possibility of preventing or treating the disease. dramatically enhances That is, it is possible to prevent or treat fibrosis occurring in each tissue caused by a decrease in tissue regeneration/repair ability caused by environmental factors such as aging and smoking.
- composition of the present invention can safely and effectively cope with fibrotic diseases, which are increasing every year along with the global aging trend, because of the low side effects of conventional fibrotic disease treatment drugs.
- the reagent composition of the present invention can be used in the study of various diseases related to cell fibrosis to help identify the mechanism of the disease and develop a preventive or therapeutic composition for the disease.
- Figure 1a is a protein band photograph of Western blot showing the anti-fibrotic action of the composition of the present invention on skin fibrosis using normal human dermal fibroblast (NHDF) as a sample.
- NHDF normal human dermal fibroblast
- the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone are shown as Western blot band intensities.
- Figure 1b is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of Figure 1a.
- Figure 2a is an experimental plan for evaluating the anti-fibrotic action of the composition of the present invention on liver fibrosis using human hepatic stellate cells (HHSC) as a sample.
- HHSC human hepatic stellate cells
- FIG. 2b is a photograph of Western blot bands showing the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone according to the experimental plan of FIG. 2a.
- Figure 2c is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of FIG. 2a.
- Figure 3a is an experimental plan for evaluating the anti-fibrotic action of the composition of the present invention on intestinal fibrosis using a human colon fibroblast cell line (CCD-18Co) as a sample.
- CCD-18Co human colon fibroblast cell line
- Figure 3b is a Western blot band photograph showing the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone according to the experimental plan of Figure 3a.
- Figure 3c is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of Figure 3b.
- Figure 4a is an experimental plan for evaluating the antifibrotic action of the composition of the present invention on cardiac fibrosis using human cardiac microvascular endothelial cells (HCMEC) as a sample.
- HCMEC human cardiac microvascular endothelial cells
- FIG. 4b is a photograph of Western blot bands showing the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone according to the experimental plan of FIG. 4a.
- Figure 4c is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of Figure 4b.
- Figure 5a is a Western blot band photograph showing the anti-fibrotic action of the composition of the present invention on skin fibrosis using normal human lung fibroblasts (NHLF) as a sample.
- NHLF normal human lung fibroblasts
- the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone are shown as Western blot band intensities.
- Figure 5b is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of Figure 5a.
- Figure 6a is an experimental plan for evaluating the fibrosis preventing action of the composition of the present invention on renal fibrosis using an epithelial cell line of human kidney renal tubular cells (Human Kidney 2, HK-2) as a sample.
- Figure 6b is a Western blot band photograph showing the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone according to the experimental plan of Figure 6a.
- Figure 6c is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensities of the western blot bands of Figure 6b.
- Figure 7a is an experimental plan for evaluating the antifibrotic action of the composition of the present invention on renal fibrosis using an epithelial cell line of human kidney renal tubular cells (Human Kidney 2, HK-2) as a sample.
- FIG. 7b is a photograph of western blot bands showing the expression levels of ⁇ SMA and GAPDH proteins according to the composition of the present invention and various concentrations of pirfenidone according to the experimental plan of FIG. 7a.
- FIG. 7c is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention and pirfenidone by quantifying the intensity of the western blot band of FIG. 7b.
- 8a is an experimental plan for evaluating the efficacy of rhHAPLN1 in changing the fibrotic morphology of HK-2 cells by TGF ⁇ 1 induction using an epithelial cell line of human kidney renal tubular cells (Human Kidney 2, HK-2) as a sample.
- FIG. 8B is a photomicrograph showing changes in shape of cells according to various concentrations of the composition of the present invention and pirfenidone according to the experimental plan of FIG. 8A.
- Figure 9a is a Western blot band photograph showing the anti-fibrotic action of the composition of the present invention on fibrosis caused by senescence, using Renal Proximal Tubule Epithelial Cells (RPTEC) as a sample.
- RPTEC Renal Proximal Tubule Epithelial Cells
- the expression levels of ⁇ SMA and GAPDH proteins according to various concentrations of the composition of the present invention for cultured cells of passage 11 are shown as Western blot band intensities.
- Figure 9b is a graph showing the relative levels of ⁇ SMA according to various concentrations of the composition of the present invention by quantifying the intensity of the western blot band shown in Figure 9a.
- 10a shows the cell nucleus observed by staining with DAPI (4',6-diamidino-2-phenylindole) during the 4th and 11th passage cultures, and ⁇ SMA antibody (abcam, ab7817) at 1:1000 in 1XPBST (1X PBS with 0.1% Triton X-100, 1% BSA) to stain intracellular ⁇ SMA, and this is a photograph showing the degree of expression by fluorescence.
- DAPI 4,6-diamidino-2-phenylindole
- FIG. 10B is a photograph showing the relative levels of ⁇ SMA in cells by fluorescence according to various concentrations of the composition of the present invention and treatment with pirfenidone during culture of 4 passages and 11 passages in which senescence was induced as a control group.
- Figure 11a is an experiment confirming the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel.
- the composition of the present invention (rhHAPLN1) showed significant effects on ⁇ SMA and collagen I at each concentration. It is a graph showing the reduction effect as a relative fold (fold change) compared to the vehicle control group.
- Figure 11b is an experiment confirming the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel.
- the composition of the present invention (rhHAPLN1) significantly reduced collagen I for each concentration It is a graph showing the effect as a relative fold compared to the vehicle control group.
- Figure 11c is an experiment confirming the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel, and the composition of the present invention (rhHAPLN1) in myofibroblasts showed a significant reducing effect on collagen IV at each concentration compared to the vehicle control group. It is a graph expressed as a relative multiple.
- Figure 12a is a diagram illustrating the outline of an experiment to evaluate the anti-fibrotic efficacy (preventive ability of fibrotic diseases) of the composition of the present invention using a mouse bleomycin-induced pulmonary fibrosis (BIPF) model.
- BIPF mouse bleomycin-induced pulmonary fibrosis
- Figure 12b is divided into four groups (Mouse 1 to 4) in the experiment using the mouse Bleomycin-induced pulmonary fibrosis model of Figure 12a normal group (Normal) group (Normal) is treated with PBS, the control group (Control) is treated with no treatment 0.0005% (w/w) of the composition of the present invention (rhHAPLN1) and 0.0015% (w/w) of the composition of the present invention (rhHAPLN1) were respectively treated and the lung tissues of 4 mice per group were extracted. Next, the left large lobe was cut in half horizontally, the upper part was fixed in formalin, and hematoxylin and eosin (H&E) staining was performed.
- Normal normal group
- Control Control
- rhHAPLN1 the composition of the present invention
- rhHAPLN1 0.0015%
- 12c shows that the middle part of the largest lobe lung tissue of the left mouse was cut to make 3 tissue slides per mouse, and 3 pictures were taken at random on each slide, and then the stained red part and the other 9 parts were taken. It is a graph confirming statistical significance by obtaining an average value per animal by averaging each value obtained by measuring the area of the stained red part using 'Image J' software.
- 12d and 12e are photographs and descriptions of the Ashcroft score, which provides an official guideline that can easily measure the severity of pulmonary fibrosis with the naked eye (Ashcroft et al 1988, J Clin Pathol 41:467-470).
- Figure 12f is a graph of the experimental results showing the severity of pulmonary fibrosis as Ashcroft score according to Figures 13a and 13b.
- Figure 13a is a diagram illustrating the outline of an experiment to evaluate the anti-fibrotic efficacy (therapeutic ability of fibrotic diseases) of the composition of the present invention using a mouse bleomycin-induced pulmonary fibrosis (BIPF) model.
- BIPF mouse bleomycin-induced pulmonary fibrosis
- Figure 13b shows mice in four groups (Normal, PBS Control, 0.00075% (w / w) rhHAPLN1, 0.0015% (w / w) rhHAPLN1, 0.003% ( w/w) rhHAPLN1), set to 4 mice per group, and after the experiment, the lung tissue of 4 mice per group was extracted, and then the left large lobe was cut in half horizontally, the upper part was fixed in formalin, and hematoxin and eosin ( H&E) This is a photograph of staining.
- H&E hematoxin and eosin
- 13c and 13d show that the middle part of the largest lobe lung tissue of the left mouse was cut to make 3 tissue slides per animal, and 3 pictures were taken at random on one slide, respectively, and the red part stained for a total of 9 parts It is a graph confirming the statistical significance by dividing the white color and the non-white color, and obtaining the average value per animal by averaging each value obtained by measuring the area of the stained red part using 'Image J' software.
- FIG. 13e is a graph of experimental results showing the severity of pulmonary fibrosis as an Ashcroft score in relation to the results of FIG. 13c.
- Figure 14a is an experimental plan for evaluating the anti-fibrotic action of the composition of the present invention on renal fibrosis using ischemia/reperfusion as an animal model for inducing renal fibrosis.
- Figure 14b is a protein band photograph of Western blot showing the expression level of ⁇ SMA protein according to the administration of the composition of the present invention and pirfenidone according to the experimental plan of Figure 14a (SV; sham-vehicle, SB; sham-rhHAPLN1-dissolving Sham-rhHAPLN1 dose B as a solution, group to check the effect of rhHAPLN1 treatment on Sham control, IRV; IR-vehicle, IRP; IR-pirfenidone, IRA; IR-rhHAPLN1 dose A, IRB; IR- rhHAPLN1 dose B, IRC; IR-rhHAPLN1 dose C).
- SV sham-vehicle, SB
- sham-rhHAPLN1-dissolving Sham-rhHAPLN1 dose B as a solution, group to check the effect of rhHAPLN1 treatment on Sham control
- IRV
- FIG. 14c is a graph showing the expression level of ⁇ SMA against IRV as a fold change according to various concentrations of pirfenidone and the composition of the present invention by quantifying the intensity of the Western blot band of FIG. 14b.
- 14D is a photograph showing the expression level of Collagen according to various concentrations of pirfenidone and the composition of the present invention in a Sirus red and PAS staining photograph in an acute renal injury-induced model. Purple color shows collagen expression.
- FIG. 14e shows two random sites in the outer medulla of the kidney in tissue staining to quantify the expression level of Collagen according to various concentrations of pirfenidone and the composition of the present invention in the acute kidney injury induced model. This is a picture showing only the collagen-positive area after imaging with .
- FIG. 14f is a graph shown by marking collagen-positive sites in FIG. 14e and quantifying the area (%) of collagen using i-solution software (IMT).
- IMT i-solution software
- 14g is a graph showing values of creatinine clearance measured at 21 days after ischemia/recanalization according to various concentrations of pirfenidone and the composition of the present invention in an acute renal injury-induced model.
- rhHAPLN1 represents recombinant human HAPLN1 as an abbreviation for recombinant human HAPLN1.
- fibroblast refers to cells involved in maintaining tissue structures by making connective tissues such as collagen fibers, not epithelial, blood vessels, lymph vessels, and inflammatory cells.
- Myofibroblast refers to fibroblasts activated by physical injury or inflammation and having a contractile function like smooth muscle cells by expressing ⁇ -SMA (alpha-smooth muscle actin).
- ⁇ SMA or " ⁇ -SMA (alpha smooth muscle actin)
- ⁇ SMA alpha smooth muscle actin
- ⁇ SMA is a protein expressed in pathological vascular smooth muscle cells and stromal fibroblastic cells, It is a major marker of tissue fibrosis. Expression is high in the kidney showing findings of renal fibrosis, and such a high expression level or activity means that the activity of myofibroblasts, which is closely related to the degree of interstitial fibrosis, is high.
- ⁇ SMA since the expression of ⁇ SMA in the patient is closely related to the decrease in creatine clearance in the kidney, it functions as a marker that can confirm that creatine, a waste product in the body, is not cleared.
- transforming growth factor beta 1 refers to a cytokine used to transform fibroblasts into myofibroblasts in various embodiments of the present invention, and the conversion capacity for each cell is different
- various physiologically active substances such as cytokines, chemokines, growth factors, and hormones are involved in the transformation of fibroblasts into myofibroblasts.
- Pirfenidone is an anti-fibrotic drug that has been approved as a treatment for pulmonary fibrosis and is currently undergoing clinical trials as a treatment for renal fibrosis. It was used as a control material in the experiments of the examples of the present invention. Although the effective dose may be different for each cell, it has been reported to show efficacy from 100 ⁇ g/mL or more in related literature.
- One aspect of the present invention relates to an anti-fibrotic composition
- an anti-fibrotic composition comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient. It can function as a pharmaceutical composition for preventing or treating fibrotic diseases.
- HPLN1 proteoglycan link protein 1
- the recombinant human HAPLN1 protein represented by SEQ ID NO: 1 in the composition of the present invention may be used.
- the HAPLN1 protein of the present invention is 80%, preferably 85%, more preferably 90%, still more preferably 95%, most preferably 95% of the amino acid sequence of SEQ ID NO: 1 as long as it maintains the anti-fibrotic function. may be a protein having 100% sequence identity.
- the nucleic acid related to the gene encoding the HAPLN1 protein in the composition of the present invention may be included in the composition by being included in an expression vector.
- the cells to which the composition of the present invention acts are not particularly limited, but preferably include various fibroblasts, astrocytes, endothelial cells and epithelial cells, and more specifically, skin fibroblasts, liver Astrocytes, human colon cells, microvascular endothelial cells, lung fibroblasts, kidney renal tubular cells, and proximal tubular epithelial cells, etc., including cells in various situations such as in vivo, in vitro, ex vivo, and in situ.
- the organ to be applied is not particularly limited as long as it is an organ that is likely to induce fibrosis, but preferably includes the lung, kidney, skin, liver, intestine, heart, and the like.
- the composition of the present invention has a single dose to cells of 0.1 ng/ml to 500 ng/ml, preferably 1 ng/ml to 300 ng/ml, more preferably 3.0 ng/ml to 50 ng/ml, more preferably 10 ng/ml to 13 ng/ml. However, it may be more specifically 3, 5, 11, 30, 50, or 100 ng/ml depending on the area to be applied or in some cases.
- the composition of the present invention is 0.00001 to 0.1% (w / w), preferably 0.0001 to 0.05% (w / w), more preferably 0.0003 to 0.03% (w/w), still more preferably 0.0005 to 0.015% (w/w).
- the composition of the present invention may be included as a main or auxiliary active ingredient of the composition for preventing or inhibiting the fibrosis of cells.
- hyaluronan and proteoglycan link protein 1 HPLN1
- a composition comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, preventing or inhibiting cell fibrosis It's about how.
- kits for preventing or inhibiting fibrosis of cells comprising the composition and treatment instructions of the present invention.
- the present invention relates to an experimental reagent composition related to the prevention or inhibition of cell fibrosis, including the composition of the present invention.
- the present invention relates to the use of the composition of the present invention for use in the preparation of a pharmaceutical composition for preventing or treating diseases caused by cell fibrosis.
- the pharmaceutical composition may generally include a molecule and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes saline solutions, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds may also be included in the composition.
- the pharmaceutical composition of the present invention may further include a pharmaceutical additive selected from the group consisting of a pharmaceutically acceptable carrier, diluent, binder, disintegrant, lubricant, and any combination thereof.
- the pharmaceutical composition may be formulated to be compatible with the intended route of administration.
- the composition of the present invention may be a formulation selected from the group consisting of eye drops, ointments, tablets, pills, capsules, troches, inhalants, injections, patches, and suppositories.
- routes of administration include parenteral, eg intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal and rectal administration. It is preferably for parenteral administration.
- Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate and tonicity adjusting agents such as sodium chloride or dextrose.
- the pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
- Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the composition must be sterile and must be fluid enough to facilitate syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersion, and the use of a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
- isotonic agents eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
- preferred methods of preparation are vacuum drying and freeze drying, resulting in the powder of the active ingredient and any additional ingredients desired from a previously sterile filtered solution.
- Oral compositions generally include an inert diluent or an edible carrier.
- the active compounds may be incorporated with excipients and used in the form of tablets, troches or capsules, eg gelatin capsules.
- Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
- Pharmaceutically compatible binders and/or adjuvant materials may be included as part of the composition.
- Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch
- lubricants such as magnesium stearate or sterotes
- glidants such as colloidal silicon dioxide
- sweeteners such as sucrose or saccharin
- a flavoring such as peppermint
- the compounds are delivered in the form of an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant for example a gas such as carbon dioxide, or a nebulizer.
- Systemic administration may also be by transmucosal or transdermal means.
- penetrants suitable for the barrier to be penetrated are used in the formulation.
- penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
- Transmucosal administration can be carried out using nasal sprays or suppositories.
- the active compounds are formulated into ointments, salves, gels or creams as is generally known in the art.
- the compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- suppositories eg, with conventional suppository bases such as cocoa butter and other glycerides
- retention enemas for rectal delivery.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage can vary within this range depending on the dosage form used and the route of administration used.
- a therapeutically effective amount (ie effective dosage) of the composition of the present invention will depend on the circumstances of the selected patient. For example, a single dose ranging from about 1 pg to 1000 mg may be administered; In some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg may be administered. there is.
- the dose of rhHAPLN1 protein is 0.001 to 10 mg/kg/BW, preferably 0.004 to 5 mg/kg/BW. , more preferably 0.1 to 1.0 mg/kg/BW, still more preferably 0.15 to 0.5 mg/kg/BW.
- composition may be administered.
- the pharmaceutical composition may be administered from one or more times a day to one or more times a week, including once every other day. Skilled artisans will recognize that certain factors may influence the dosage and timing required to effectively treat a subject, including the severity of the disease or disorder, previous treatments, general health and/or age of the subject, and other pre-existing factors. diseases, including but not limited to.
- treatment of a subject with a therapeutically effective amount of a molecule of the invention may include a single treatment or, preferably, may include a series of treatments.
- the dosage of the composition of the present invention is 5 mg/kg/week to 500 mg/kg/week, for example 5 mg/kg/week, 10 mg/kg/week, 15 mg/kg/week, depending on the patient's circumstances. , 20 mg/kg/week, 25 mg/kg/week, 30 mg/kg/week, 35 mg/kg/week, 40 mg/kg/week, 45 mg/kg/week, 50 mg/kg/week, 55mg/kg/week, 60mg/kg/week, 65mg/kg/week, 70mg/kg/week, 75mg/kg/week, 80mg/kg/week, 85mg/kg/week, 90mg/kg/week, 95mg/week kg/week, 100mg/kg/week, 150mg/kg/week, 200mg/kg/week, 250mg/kg/week, 300mg/kg/week, 350mg/kg/week, 400mg/kg/week, 450mg/kg/ weeks and 500 mg/kg/week.
- the dosage of the bifunctional molecule according to the present invention is 10 mg/kg/week to 200 mg/kg/week, 20 mg/kg/week to 150 mg/kg/week or 25 mg/kg/week. weeks to 100 mg/kg/week.
- a composition of the present invention is administered for 2 weeks to 6 months, for example 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks. weeks, 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or 1x administration per week for longer than 1 year.
- the composition of the present invention is administered 2x times per week.
- the composition of the present invention is administered every other week.
- composition of the present invention can also be formulated as a pharmaceutical composition comprising a pharmacologically effective amount of the HAPLN1 protein molecule and a pharmaceutically acceptable carrier.
- a pharmacologically or therapeutically effective amount means an amount effective to produce the intended pharmacological, therapeutic or prophylactic result.
- the phrases "pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to an amount of a bifunctional molecule effective to produce the intended pharmacological, therapeutic or prophylactic result. For example, if a given clinical treatment is considered effective when it reduces a measurable parameter associated with a disease or disorder by at least 20%, then a therapeutically effective amount of a drug to treat that disease or disorder will reduce that parameter by at least 20%. is the amount needed to reduce
- compositions of the present invention can be administered by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. Administration can be by any means known in the art.
- the pharmaceutical composition is administered by intravenous or parenteral infusion or injection.
- a suitable dosage unit of the molecule is in the range of 0.001 to 0.25 mg per kilogram of body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram of body weight per day, or in the range of 0.01 to 10 micrograms per kilogram of body weight per day; or in the range of 0.10 to 5 micrograms per kilogram of body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram of body weight per day.
- a pharmaceutical composition comprising the molecule may be administered once daily. However, the therapeutic agent may also be administered in dosage units containing 2, 3, 4, 5, 6 or more sub-doses administered at appropriate intervals throughout the day.
- Dosage units may also be formulated into a single dose over several days using, for example, conventional sustained release formulations that provide sustained and consistent release of the molecule over a period of several days. Sustained release formulations are well known in the art.
- the dosage unit contains multiples corresponding to the daily dosage.
- the pharmaceutical composition may be included in a kit, container, pack, or dispenser along with instructions for administration.
- treatment means to treat, cure, alleviate, alleviate, alter, relieve, ameliorate, ameliorate, or affect a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease. It is defined as the application or administration of a therapeutic agent (e.g., a molecule of the invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, wherein the patient has a disease or disorder, a symptom of the disease or disorder, or predisposition to a disease or disorder.
- a therapeutic agent e.g., a molecule of the invention
- one aspect of the present invention relates to the use of a composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) as an active ingredient to prevent or inhibit fibrosis of cells.
- HPLN1 hyaluronan and proteoglycan link protein 1
- HPLN1 hyaluronan and proteoglycan link protein 1
- a CHO-K1 cell line producing recombinant human HAPLN1 protein was constructed by inserting a vector containing a polynucleotide encoding the human HAPLN1 protein of amino acid sequence number 1 into CHO-K1 cells.
- amino acid sequence number 1 is as follows:
- MCB Master Cell Bank
- the MCB was subcultured and inoculated into Thermo's Hyperforma SUB 250 L bioreactor at a concentration of 0.40 ⁇ 0.05 ⁇ 10 6 cells/mL, followed by fed-batch culture.
- As a basic medium 22.36 g ActiProTM medium + 0.5846 g glutamine + 10.00 g HT Supplement (Thermo Fisher Scientific) + 4.29 g 10 N NaOH + 1.80 g NaHCO 3 was used.
- the culture temperature was set at 36.5°C, dissolved oxygen (DO) at 40.0%, and pH at 7.00 ⁇ 0.20. 1 M sodium carbonate monohydrate was used as a pH control solution.
- FM020a 181.04 g HyCloneTM Cell Boost 7a + 12.28 g 10 N NaOH and 94.60 g HyCloneTM Cell Boost 7b + FM020b of 105.93 g 10 N NaOH were used.
- rhHAPLN1 recombinant human HAPLN1 (rhHAPLN1) protein from cells cultured as above (Harvest and Clarification), ultrafiltration/diafiltration 1 (UF/DF1), anion exchange chromatography, S/D (Solvent/Detergent ) Virus Inactivation, Cation Exchange Chromatography, Mixed-mode Chromatography (MMC), Hydrophobic Interaction Chromatography, Ultrafiltration/Diafiltration 2 (UF/DF2) and Medium Depth Filtration ( It was separated and purified through processes such as Intermediate Depth Filtration, Int. DF).
- the composition of the present invention was used.
- composition of the present invention containing the HAPLN1 protein
- the HAPLN1 protein purified in Preparation Example 1 was stabilized in 20 mM acetate buffer, 8% (w/v) sucrose, 0.04% (w/v) PS80, pH 5.0 to contain the HAPLN1 protein itself as the main active ingredient.
- a composition of the invention was prepared.
- composition of the present invention containing an expression vector containing the gene encoding the HAPLN1 protein
- a composition containing an expression vector containing the gene encoding the HAPLN1 protein was prepared using the jetPRIME Transfection Reagent Kit (114-15, PolyPlus). More specifically, a plasmid vector designed to express Human HAPLN1 was prepared using OriGene's RC209274 product (hereinafter referred to as "Human HAPLN1 ORF Clone"). It can express the genes it transports in the host cell's nucleus.
- the jetPRIME Transfection Reagent Kit consists of jetPRIME buffer and jetPRIME Transfection reagent. It works to infiltrate the inside.
- the human HAPLN1 ORF clone was diluted in jetPRIME buffer to prepare a composition of the present invention containing an expression vector containing a gene encoding the HAPLN1 protein.
- jetPRIME Transfection reagent is added and allowed to stand for 10 minutes so that the plasmid vector is captured in the form of a vesicle (liposome) and then dropped onto the cells in culture. After culturing the cells for more than 48 hours, an increase in human HAPLN1 gene expression was confirmed by real-time qPCR, and an increase in human HAPLN1 protein expression was confirmed by Western blot.
- composition of the present invention on skin fibrosis was verified using normal human dermal fibroblast (NHDF) as a sample.
- NHDF normal human dermal fibroblast
- Human dermal fibroblasts Normal Human Dermal Fibroblast, NHDF
- FBM-2 Fibroblast Growth Medium-2, CC-3131, Lonza
- composition of the present invention was applied to each well of the plate under conditions in which fibrosis was induced by treatment with TGF ⁇ 1 (10 ng/ml), but rhHAPLN1 protein content of 0, 3, 10, 30, and After treatment with 100 ng/ml and commercially available anti-fibrotic agent pirfenidone (Selleckchem) 100 ⁇ g/mL and 200 ⁇ g/mL, respectively, the cells were incubated for 24 hours.
- FIG. 1a it can be seen from the Western blot band photograph that the composition of the present invention exhibits a much better anti-fibrotic action than pirfenidone, considering the thickness and thickness of the ⁇ SMA protein band with respect to the degree of fibrosis of normal human dermal fibroblasts. there was.
- the expression level of ⁇ SMA was significant in all rhHAPLN1 protein content ranges when treated with the composition of the present invention, compared to when treated with a composition without rhHAPLN1 protein after inducing fibrosis with TGF1 ⁇ (10 ng/ml). showed low anti-fibrotic efficacy.
- the sample with rhHAPLN1 protein content of 100 ng/ml showed the best anti-fibrotic efficacy, which is 1/1000 of the content of pirfenidone 100 ⁇ g/mL (100,000 ng/ml). 25% higher results.
- pirfenidone since the expression level of ⁇ SMA is higher at 200 ⁇ g/mL than at 100 ⁇ g/mL, it can be assumed that the anti-fibrotic efficacy at high concentration is rather low and 100 ⁇ g/mL is the optimal anti-fibrotic concentration. Then, it was found that the composition of the present invention has much better antifibrotic efficacy than pirfenidone, a commercially available antifibrotic agent.
- composition of the present invention on liver fibrosis was verified using human hepatic stellate cells (HHSC) as a sample.
- HHSC human hepatic stellate cells
- each well of the plate was treated with the composition of the present invention with rhHAPLN1 protein content of 0, 3, 10, 30, and 100 ng/ml and pirfenidone 100 ⁇ g/mL and 200 ⁇ g/mL, respectively, for 24 hours incubated.
- each sample was treated with a cell lysis buffer and harvested.
- samples with a rhHAPLN1 protein content of 3 ng/ml showed the best anti-fibrotic efficacy, which was demonstrated by pirfenidone 100 ⁇ g/mL (100000 ng/ml) as well as pirfenidone 200 ⁇ g/mL (200000 ng/ml), despite the content being 3/200000, the anti-fibrotic efficacy showed much higher results.
- composition of the present invention on intestinal fibrosis was verified using human colon fibroblast cell line (CCD-18Co) as a sample.
- human colon fibroblast cell line (CCD-18Co (human colon fibroblast cell line; ATCC, CRL-1459)) was seeded in a 6-well plate (1.5 X 10 5 cells/well). ) and EMEM (Eagle's minimum essential medium, ATCC) medium containing 10% fetal bovine serum (FBS) was incubated for 24 hours in an incubator at 37°C and 5% CO 2 condition.
- CCD-18Co human colon fibroblast cell line
- ATCC human colon fibroblast cell line
- CRL-1459 human colon fibroblast cell line
- FBS fetal bovine serum
- each well of the plate was treated with the composition of the present invention with rhHAPLN1 protein content of 0, 3, 10, 30, and 100 ng/ml and pirfenidone 100 ⁇ g/mL and 200 ⁇ g/mL, respectively, for 24 hours incubated.
- Figure 3b shows that the composition of the present invention exhibits much better anti-fibrotic activity than pirfenidone in all rhHAPLN1 protein content ranges, as seen from the thickness and thickness of the ⁇ SMA protein band with respect to the degree of fibrosis of human colonic fibroblasts. Western blot This could be confirmed from the band photos.
- myofibroblasts (myofibroblasts) were significantly reduced in all rhHAPLN1 protein content ranges when treated with the composition of the present invention, compared to the case where fibrosis was induced with TGF ⁇ 1 (10 ng/ml) and then treated with a composition that did not contain rhHAPLN1 protein. )
- the expression level of ⁇ SMA as a marker was low, showing anti-fibrotic efficacy.
- intestinal fibrosis myofibroblasts are transformed into myofibroblasts to produce excessive fibrous tissue.
- samples with rhHAPLN1 protein content of 10 ng/ml showed the best anti-fibrotic efficacy, which was induced by TGF ⁇ 1 even though the content was 1/10000 of that of pirfenidone 100 ⁇ g/mL (100,000 ng/ml).
- the antifibrotic efficacy is about 7 times higher when comparing the efficacy of drugs, and the antifibrotic efficacy is much higher than 2 times even though the content is 1/20000 of pirfenidone 200 ⁇ g/mL (200000 ng/ml) .
- pirfenidone since the expression level of ⁇ SMA is higher at 200 ⁇ g/mL than at 100 ⁇ g/mL, it can be assumed that the anti-fibrotic efficacy at high concentrations is rather low, and that 100 ⁇ g/mL is the optimal anti-fibrotic concentration of pirfenidone. Then, it can be seen that the composition of the present invention has excellent antifibrotic efficacy compared to pirfenidone, which is a commercially available antifibrotic agent.
- HCMEC human cardiac microvascular endothelial cells
- HCMEC human cardiac microvascular endothelial cells
- ScienCell #6000
- each well of the plate was treated with the composition of the present invention with rhHAPLN1 protein content of 0, 3, 10, 30, and 100 ng/ml and pirfenidone 100 ⁇ g/mL and 200 ⁇ g/mL, respectively, for 2 days incubated.
- each sample was treated with a cell lysis buffer and harvested.
- Figure 4b shows that the composition of the present invention exhibits much better anti-fibrotic activity than pirfenidone in all rhHAPLN1 protein content ranges, in terms of the thickness and thickness of the ⁇ SMA protein band for the degree of fibrosis of human cardiac microvascular endothelial cells. It could be confirmed from the Western blot band picture.
- myofibroblasts (myofibroblasts) were significantly reduced in all rhHAPLN1 protein content ranges when treated with the composition of the present invention, compared to when fibrosis was induced with TGF ⁇ 1 (5 ng/ml) and then treated with a composition not containing rhHAPLN1 protein. )
- the expression level of ⁇ SMA as a marker was low, showing anti-fibrotic efficacy.
- endothelial cells are transformed into myofibroblasts to produce excessive fibrous tissue.
- compositions of the present invention a sample with a rhHAPLN1 protein content of 3 ng/ml showed the best anti-fibrotic efficacy, which was 3/100,000 higher than that of 100 ⁇ g/mL (100,000 ng/ml) of pirfenidone.
- An antifibrotic effect that was not shown was shown, and an antifibrotic effect that was not shown in pirfenidone was shown despite the content of 3/200000 of pirfenidone 200 ⁇ g/mL (200000 ng/ml).
- composition of the present invention can be an almost unique antifibrotic agent, at least for cardiac microvascular endothelial cells.
- composition of the present invention on pulmonary fibrosis was verified using normal human lung fibroblast (NHLF) as a sample.
- NHLF normal human lung fibroblast
- NHLF normal human lung fibroblasts
- composition of the present invention was administered to each well of the plate under conditions in which fibrosis was induced by treatment with TGF ⁇ 1 (10 ng/ml), rhHAPLN1 protein content of 0, 3.1, 12.5, 25, and 100 ng/ml and Pirfeni.
- Pigs (Selleckchem) were treated with 100 ⁇ g/mL and 200 ⁇ g/mL, respectively, and then incubated for 24 hours.
- Figure 5a shows that the composition of the present invention exhibits a much better anti-fibrotic action than pirfenidone in all rhHAPLN1 protein content ranges, as seen from the thickness and thickness of the ⁇ SMA protein band for the degree of fibrosis of normal human lung fibroblasts. Western It could be confirmed from the blot band picture.
- myofibroblasts (myofibroblasts) were significantly reduced in all rhHAPLN1 protein content ranges when treated with the composition of the present invention, compared to when fibrosis was induced with TGF ⁇ 1 (10 ng/ml) and then treated with a composition without rhHAPLN1 protein.
- the expression level of ⁇ SMA as a marker was low, showing anti-fibrotic efficacy.
- the sample with rhHAPLN1 protein content of 12.5 ng/ml showed the best anti-fibrotic efficacy, which is 1/10,000 higher than that of pirfenidone 100 ⁇ g/mL (100,000 ng/ml). It is about 2 times higher, and it was also about 2 times higher for pirfenidone 200 ⁇ g/mL (200000 ng/ml).
- pirfenidone has no antifibrotic effect, at least in lung fibroblasts, since there is almost no difference in the expression level of ⁇ SMA compared to the case of treatment with a composition not containing rhHAPLN1 protein even when treated at any concentration.
- the composition of the present invention can be an almost unique anti-fibrotic agent for lung fibroblasts.
- the fibrosis preventive action of the composition of the present invention on renal fibrosis was verified using an epithelial cell line of human kidney renal tubular cells (Human Kidney 2, HK-2) as a sample.
- TGF ⁇ 1 (5 ng/ml) and the composition of the present invention (rhHAPLN1 protein content of 0, 5, 10, 20, and 50 ng/ml) and pirfenidone 0.2 were added to each well of the plate. They were treated simultaneously with mg/mL (200 ⁇ g/mL) and then incubated for 24 hours.
- the primary antibody was diluted 1:1000 in 1% BSA-TBST and reacted overnight at 4°C.
- the secondary antibody was diluted 1:2000 in 1% BSA-TBST (Bovine Serum Albumin-Tris-Buffered Saline & Polysorbate 20), dispensed, and shaken for about 1 hour at room temperature.
- BSA-TBST Bovine Serum Albumin-Tris-Buffered Saline & Polysorbate 20
- the composition of the present invention is superior to pirfenidone in a specific rhHAPLN1 protein content range (rhHAPLN1 protein content of 50 ng/ml) in terms of the thickness and thickness of the ⁇ SMA protein band for the degree of fibrosis of human renal tubular cells. It was confirmed from the Western blot band picture that the fibrosis preventive action was exhibited.
- composition of the present invention on renal fibrosis was verified using an epithelial cell line of human renal tubular cells (Human Kidney 2, HK-2) as a sample.
- human renal tubular cells (HK-2) were seeded (2.8 X 10 5 cells/well) in a 60-well plate, and RPMI 1640 Medium (11875 -093, Gibco) in an incubator at 37° C. 5% CO 2 conditions for 24 hours.
- composition of the present invention rhHAPLN1 protein content of 0, 5, 10, 20, and 50 ng/ml
- 0.2 mg/mL (200 ⁇ g/mL) of pirfenidone were incubated for 48 hours.
- the secondary antibody was diluted 1:2000 in 1% BSA-TBST and shaken for about 1 hour at room temperature.
- the composition of the present invention has an anti-fibrotic activity in a specific rhHAPLN1 protein content range (rhHAPLN1 protein content of 10 to 50 ng / ml), considering the thickness and thickness of the ⁇ SMA protein band for the degree of fibrosis of human renal tubular cells. It could be confirmed from the Western blot band picture.
- the composition of the present invention when fibrosis was induced with TGF- ⁇ (10 ng/ml) and then treated with a composition without rhHAPLN1 protein, the composition of the present invention was treated with rhHAPLN1 protein content of 10 to 50 ng/ml. , especially when treated with rhHAPLN1 protein content of 50 ng/ml, it showed excellent antifibrotic efficacy. It is quite noteworthy that the treatment with 50 ng/ml of the composition of the present invention showed antifibrotic efficacy almost equivalent to that of pirfenidone, even though the amount was 1/4 smaller than 0.2 mg/ml of pirfenidone, that is, 200 ng/ml. .
- composition of the present invention on renal fibrosis was verified by observing changes in cell morphology using an epithelial cell line of human kidney renal tubular cells (Human Kidney 2, HK-2) as a sample.
- Human kidney renal tubular cells (HK-2) were seeded (3.0 X 10 5 cells/well) in a 6-well plate according to the experimental plan of FIG. 8a and supplemented with 10% FBS in RPMI 1640 Medium (11875 -093, Gibco) in an incubator at 37° C. 5% CO 2 conditions for 24 hours.
- composition of the present invention (rhHAPLN1 protein content of 0, 5, 10, 20, 50 and 100 ng/ml) and pirfenidone 0.2 mg/mL (200 ⁇ g/mL) were respectively treated and incubated for 24 hours. .
- the shape of the cell is shown in various ways depending on the treated material.
- the control group which is a normal cell without any treatment
- the cell shape was round, but it was confirmed that the round cell shape changed to a fibrotic form by 10ng/ml TGF ⁇ 1 treatment.
- composition of the present invention having various rhHAPLN1 protein contents was added thereto, the shape of the cells was changed to round as in the case of pirfenidone.
- composition of the present invention clearly exhibited excellent anti-fibrotic efficacy against cells.
- the antifibrotic activity of the composition of the present invention against renal fibrosis due to aging induction was verified using primary epithelial cells of renal proximal tubule epithelial cells (RPTEC) as samples.
- RPTEC renal proximal tubule epithelial cells
- Renal proximal tubular epithelial cells (RPTEC) of passage 3 and passage 10 were seeded (5.0 X 10 5 cells) in a 60 ⁇ culture dish, and complete Renal Epithelial Cell Medium (ATCC PCS-400-030+ATCC PCS-400-040) in an incubator at 37°C and 5% CO 2 conditions for 24 hours.
- RPTEC Renal proximal tubular epithelial cells
- each well was treated with 300 ⁇ l of RIPA Lysis and Extraction Buffer containing a protease inhibitor and a phosphatase inhibitor, and the cells were recovered by scraping with a scraper and transferred to an E-tube.
- the antifibrotic activity of the composition of the present invention against renal fibrosis due to aging induction was verified using primary epithelial cells of renal proximal tubule epithelial cells (RPTEC) as samples.
- RPTEC renal proximal tubule epithelial cells
- Renal proximal tubular epithelial cells (RPTEC) of 3 and 10 passages were seeded (1.5 X 10 5 cells) in a 12-well plate, and complete Renal Epithelial Cell Medium (ATCC PCS-400-030+ATCC PCS-400-040) in an incubator at 37°C and 5% CO 2 conditions for 24 hours.
- the composition of the present invention is It can be seen that it exhibited excellent antifibrotic efficacy compared to conventional antifibrotic agents.
- composition of the present invention on pulmonary fibrosis and renal fibrosis was verified by examining the expression levels of various fibrosis markers using the BioMAP Fibrosis Panel (provided by Eurofins discovery).
- the BioMAP Fibrosis panel consists of three models: SAEMyoF system (co-culturing small airway epithelial cells and adult fibroblasts), REMyoF system (co-culture of renal proximal tubule epithelial cells and adult fibroblasts), and MyoF system (differentiated lung myofibroblasts).
- SAEMyoF system represents interstitial lung diseases (IDL) including pulmonary fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), and the REMyoF system represents end-stage renal disease (end-stage renal disease). It represents kidney fibrosis disease associated with stage renal failure.
- IDL interstitial lung diseases
- IPF pulmonary fibrotic diseases
- REMyoF system represents end-stage renal disease (end-stage renal disease). It represents kidney fibrosis disease associated with stage renal failure.
- experiments were conducted to determine the effect of the MyoF system only on myofibroblasts.
- peripheral blood mononuclear cells peripheral blood mononuclear cells
- composition of the present invention (rhHAPLN1 protein content of 0, 3.7, 11, and 33 ng/ml) was treated at each concentration and incubated for 1 hour.
- the composition of the present invention (rhHAPLN1) had a significant reducing effect on ⁇ SMA and collagen I in the SAEMyoF system, which is a pulmonary fibrosis disease model (FIG. 11a). That is, in the case of ⁇ SMA, it was confirmed that the treatment with the composition of the present invention consistently reduced the expression of ⁇ SMA by 0.9 times or less, regardless of the content of rhHAPLN1 protein in the composition, compared to the treatment with the composition not containing the rhHAPLN1 protein.
- the composition of the present invention (rhHAPLN1) had a significant reducing effect on collagen IV (FIG. 11c). That is, it was confirmed that the treatment with the composition of the present invention reduced the expression level of collagen I to 0.8 times or less in proportion to the content of rhHAPLN1 protein in the composition, compared to the treatment with the composition not containing the rhHAPLN1 protein. Therefore, when the content of rhHAPLN1 protein in the composition was 33 ng/ml, the expression level of collagen IV was reduced by 0.7 times.
- mice 8-week-old female C57BL/6N (KBSI, Daejeon City, Korea) mice were used, and a normal control group (Normal, PBS) and two dose groups (composition of the present invention containing rhHAPLN1 protein) were set. Water was exchanged every two days and allowed to eat freely. Four mice were placed in one cage, and the temperature in the breeding room was maintained at 21 to 24 ° C and the humidity was maintained at 40% to 60%, and the day and night cycles were each 12 hours. .
- KBSI normal, PBS
- two dose groups composition of the present invention containing rhHAPLN1 protein
- PF pulmonary fibrosis
- idiopathic pulmonary fibrosis (IPF) was performed using a 1ml-syringe and an oral needle (Oral Zonde Needle 0.9x50mm; 20Guage). After anesthesia, 25 ⁇ l once orally intratracheal at a concentration of 2 U/kg was carefully administered (instillation), followed by fresh 10 ml phosphate-buffered saline (1X PBS, pH 7.4, Gibco Co.) was added dropwise to a nebulizer to nebulize and inhale freely.
- Control was 50 ⁇ l of a stock solution composed of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, 50% glycerol and 10 ml PBS (1X, pH 7.4, Gibco Co.
- rhHAPLN1 stock solution 50 ⁇ l rhHAPLN1 weight ratio concentration 0.0005% (w / w), that is, 5000ng / mL, and 0.0015% (w / w), that is, 15000ng
- each was mixed in 10 ml PBS (1X, pH 7.4, Gibco Co.), and administered in groups for 1 hour once a day in a nebulization manner through the above nebulizer. .
- the weight of each experimental animal was measured every 2 days to confirm that there was no change in weight, and a Mass Dosing System and Aerosol Chamber (Data Science International Co.) were used for mist generation.
- the total experimental period was 12 days, and recombinant HAPLN1 protein administration at each concentration was administered 10 times daily for 1 hour for 10 days, and dissection was performed after anesthesia on the 11th day.
- Figure 12b is a photograph of the left large lobe after lung tissue was removed, cut in half horizontally, and the upper part was fixed in formalin and stained with hematoxylin and eosin (H&E). It can be seen from the photograph that when the composition of the present invention is administered compared to the control group, in particular, in the case of rhHAPLN1 weight ratio concentration of 0.0015% (w/w), it shows a very similar form to the normal group.
- H&E hematoxylin and eosin
- the middle part of the left large lobe of the mouse was cut to make 3 tissue slides per mouse, and 3 pictures were taken at random on each slide, and then, for a total of 9 parts, the stained red part and the unstained white part were distinguished, The average value per animal was obtained by averaging each value obtained by measuring the area of the stained red part using Image J software.
- the red part represents the part where fibrosis has progressed.
- FIG. 12c which shows these average values as a graph
- the composition of the present invention when the composition of the present invention was administered compared to the control group, in particular, in the case of rhHAPLN1 weight ratio concentration of 0.0015% (w/w), the average value of the area of the red part was reduced to be considerably close to the normal group. Able to know. That is, it was confirmed that the composition of the present invention exhibits a preventive effect against pulmonary fibrosis in vivo.
- 12d and 12e are photographs and descriptions (Ashcroft et al 1988, J Clin Pathol 41:467-470) explaining official guidelines that can simply measure the severity of pulmonary fibrosis with the naked eye.
- the grade of fibrosis can be divided by the Ashcroft score as follows:
- Score 1 isolated soft fibrosis change (diaphragm thickness less than or equal to 3 times normal), alveoli partially enlarged, sparse, but no fibrotic mass (lump).
- Score 2 Clear fibrotic change (diaphragm thickness greater than 3 times normal), alveoli are partially enlarged and sparse, but no fibrotic mass is observed.
- Score 4 Changes in structure, single fibrous mass less than or equal to 10% of the microscopic area.
- Score 5 Structural changes, confluent fibrous masses (greater than 10% and less than or equal to 50% of the microscopic area), lung structures severely damaged but still retaining morphology.
- each of the three observers judged and scored individually under the condition that they were not affected by the evaluation of others with the same data, and then combined the results to determine the final average score. Results of each rater were very similar and the results of a double representative observer are shown in FIG. 12f.
- PF pulmonary fibrosis
- idiopathic pulmonary fibrosis (IPF) was performed using a 1ml-syringe and an oral needle (Oral Zonde Needle 0.9x50mm; 20Guage). After anesthesia, 20 ⁇ l per mouse at a concentration of 50 U/kg was administered orally intratracheal once (instillation), followed by fresh 10 ml phosphate-buffered saline (1X PBS, pH 7.4, Gibco Co.) was added dropwise to a nebulizer to nebulize and inhale freely.
- the control was 50 ⁇ l of a stock solution composed of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, 50% glycerol and 10 ml PBS (1X, pH 7.4, Gibco Co. ) was used as a mixture of solutions.
- the rhHAPLN1 stock solution (20 mM acetate, pH 5.0, 8.0 M sucrose, 0.04% PS80) was diluted with phosphate buffered saline (PBS) to obtain a rhHAPLN1 weight ratio concentration of 0.00075% (w /w), i.e.
- the total experimental period was 23 days, and 10 ml of recombinant human HAPLN1 protein at each concentration was administered 14 times a day for 1 hour each time for 14 days, and dissection was performed after anesthesia the day after the final exposure.
- Figure 13b is a photograph of the left large lobe after lung tissue was removed, cut in half horizontally, and the upper part was fixed in formalin and stained with hematoxylin and eosin (H&E). From the photograph, it can be seen that when the composition of the present invention is administered compared to the control group, especially in the case of rhHAPLN1 weight ratio concentration of 0.0015% (w/w), it also shows a very similar form to the normal group.
- H&E hematoxylin and eosin
- the middle part of the left large lobe of the mouse was cut to make 3 tissue slides per mouse, and 3 pictures were taken at random from each slide, and then, for a total of 9 parts, the red part that was stained and the white part that was not stained were distinguished, and Image Each value obtained by measuring the area of the stained red part using J software was averaged to obtain an average value per animal.
- the red part represents the part where fibrosis has progressed.
- FIG. 13c which shows these average values as a graph
- the composition of the present invention when the composition of the present invention was administered compared to the control group, especially in the case of rhHAPLN1 weight ratio concentration of 0.0015% (w/w), the area of the red part was compared to that of untreated lung fibrosis. It can be seen that the average value is reduced by 51%. That is, it was confirmed that the composition of the present invention exhibits an excellent therapeutic effect on pulmonary fibrosis in vivo. This is also graphed in FIG. 13D. *** p ⁇ 0.001, calculated by Student's t-test.
- mice After 1 day of ischemia, blood urea nitrogen (BUN) was measured, and only mice whose value rose to about 98 mg/dL (to be specified as 100 mg/dL in the future) or higher were grouped into experimental groups and the experiment was conducted.
- BUN blood urea nitrogen
- mice To the experimental groups, various drugs including the composition of the present invention were administered daily from the 7th day after surgery, and the kidneys were taken on the 14th day and used for histological and biochemical analysis. The drug concentration is shown in Table 1, and the details of the experimental groups are shown in Table 2.
- composition rhHAPLN1 (0.2 ⁇ g/ ⁇ l) rhHAPLN1 buffer PBS rhHAPLN1 dose A (0.1mg/kg B.W.) 12.5 ⁇ l 0 ⁇ l 187.5 ⁇ l rhHAPLN1 dose B (0.02mg/kg B.W) 2.5 ⁇ l 10 ⁇ l 187.5 ⁇ l rhHAPLN1 dose C (0.004mg/kg B.W) 0.5 ⁇ l 12 ⁇ l 187.5 ⁇ l rhHAPLN1 buffer (Vehicle) 0 ⁇ l 12.5 ⁇ l 187.5 ⁇ l Pirfenidone (300mg/kg B.W.)
- Biochemical analysis evaluation of expression of fibrosis markers: To determine the expression level of ⁇ SMA in kidney tissue, Western blot was performed using antibodies against these.
- SV sham-vehicle, SB; sham-rhHAPLN1-dissolving solution, IRV; IR-vehicle, IRP; IR-pirfenidone, IRA; IR-composition of the present invention (rhHAPLN1) A, IRB; IR-composition of the present invention (rhHAPLN1) B, IRC; IR-represents the composition (rhHAPLN1) C of the present invention, respectively, and more details are as shown in Tables 1 and 2 above. More specifically, the Sham-vehicle group was IP injected by diluting only the rhHAPLNB1-free buffer in PBS in the Sham control group.
- Sham-rhHAPLN1-dissolving solution is a group that was IP injected by diluting rhHAPLN1 concentration B (0.02mg/kg) to check the effect of rhHAPLN1 on normal conditions.
- IR-vehicle is a buffer-treated group without rhHAPLN1 in the I/R fibrosis induction model.
- IR-pirfenidone was a group in which 300 mg/kg of pirfenidone was orally administered after induction of I/R fibrosis.
- ⁇ SMA which is an indicator of fibrosis in the kidney, was significantly higher in cases of ischemia/reperfusion compared to control operations (sham-operation, SV and SB).
- ⁇ SMA in the groups (IRA and IRB) administered with the composition (rhHAPLN1) of the present invention showed a low level similar to that of pirfenidone compared to the control drug-treated group (IRV).
- IRC is considered to be high enough to be equivalent to IRV because the concentration is extremely low.
- SV sham-vehicle, SB; sham-rhHAPLN1-dissolving solution, IRV; IR-vehicle, IRP; IR-pirfenidone, IRA; IR-composition of the present invention (rhHAPLN1) concentration 0.1 mg/kg, IRB; IR-composition of the present invention (rhHAPLN1) concentration 0.02mg/kg, IRC; IR-represents the composition of the present invention (rhHAPLN1) at a concentration of 0.004 mg/kg, respectively.
- blood for this use was taken through the retroorbital vein plexus with heparinized glass capillaries, and urine was obtained using a metabolic cage.
- Blood urea nitrogen and creatinine (plasma creatinine, PCr) concentrations were measured using Vitros250 (Johnson & Johnson).
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Abstract
Description
구성 성분 | |||
rhHAPLN1 (0.2μg/μl) |
rhHAPLN1 buffer | PBS | |
rhHAPLN1 dose A (0.1mg/kg B.W) | 12.5μl | 0μl | 187.5μl |
rhHAPLN1 dose B (0.02mg/kg B.W) | 2.5μl | 10μl | 187.5μl |
rhHAPLN1 dose C (0.004mg/kg B.W) | 0.5μl | 12μl | 187.5μl |
rhHAPLN1 buffer (Vehicle) | 0μl | 12.5μl | 187.5μl |
Pirfenidone (300mg/kg B.W) |
실험군(8 weeks C57BL/6 male mouse) | 총동물수(n) | 투여방법 | |
Sham-rhHAPLN1 buffer (Vehicle) | (SV) | n=5 | Intraperitoneal injection |
Sham-rhHAPLN1 dose B (0.02mg/kg B.W) | (SB) | n=5 | Intraperitoneal injection |
IR-rhHAPLN1 buffer (Vehicle) | (IRV) | n=10 | Intraperitoneal injection |
IR-pirfenidone (300mg/kg B.W) | (IRP) | n=10 | Oral administration |
IR-rhHAPLN1 dose A (0.1mg/kg B.W) | (IRA) | n=10 | Intraperitoneal injection |
IR-rhHAPLN1 dose B (0.02mg/kg B.W) | (IRB) | n=10 | Intraperitoneal injection |
IR-rhHAPLN1 dose C (0.004mg/kg B.W) | (IRC) | n=10 | Intraperitoneal injection |
Claims (12)
- 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는, 섬유성 질환의 예방 또는 치료용 약학적 조성물.
- 청구항 1에 있어서, 상기 단백질이 서열번호 1의 아미노산 서열에 대해 80% 이상의 서열동일성을 갖는 것인 조성물.
- 청구항 1에 있어서, 상기 유전자에 관한 핵산이 발현 벡터 내에 포함되어 있는 것인 조성물.
- 청구항 1에 있어서, 상기 섬유성 질환의 병소가 피부, 간, 장, 심장, 폐, 및 신장으로 이루어지는 군에서 선택되는 것인 조성물.
- 청구항 1에 있어서, 상기 섬유성 질환의 병소가 피부 섬유아세포, 간성상 세포, 결장 섬유아세포, 심장 미세혈관 내피세포, 폐 섬유아세포, 신장 신세관 세포, 및 신장 근위 세뇨관 상피 세포로 이루어지는 군에서 선택되는 것인 조성물.
- 청구항 1에 있어서, 상기 섬유성 질환이 허혈성 섬유화증인 것인 조성물.
- 청구항 1에 있어서, 상기 조성물의 1회 투여량이 0.1ng/ml 내지 500ng/ml인 것인 조성물.
- 청구항 1에 있어서, 상기 조성물을 in vivo로 투여 시 0.001 내지 5 mg/kg BK의 rhHAPLN1 단백질의 용량으로 투여되는 것인 조성물.
- 청구항 1에 있어서, 상기 조성물이 세포의 섬유화증을 예방 또는 억제하기 위한 조성물의 주요 또는 보조 유효성분으로서 포함되는 것인 조성물.
- 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 조성물을 세포에 처리함으로써 세포의 섬유화를 예방 또는 억제하는 방법.
- 청구항 1 내지 청구항 9 중 어느 한 항에 따른 조성물 및 청구항 10의 방법에 따른 처리를 위한 지침을 포함하는 세포의 섬유화 예방 또는 억제용 키트.
- 청구항 1 내지 청구항 9 중 어느 한 항에 따른 조성물을 포함하는, 세포의 섬유화 예방 또는 억제와 관련된 실험용 시약 조성물.
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AU2022322679A AU2022322679A1 (en) | 2021-08-03 | 2022-08-02 | Composition for preventing or treating fibrotic diseases, comprising hapln1 |
CN202280054108.1A CN117881412A (zh) | 2021-08-03 | 2022-08-02 | 包括hapln1的用于预防或治疗纤维性疾病的组合物 |
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US20130052198A1 (en) | 2010-03-29 | 2013-02-28 | The General Hospital Corporation | Anti-inflammatory factors |
WO2013144349A1 (en) * | 2012-03-30 | 2013-10-03 | Societe De Developpement Et De Recherche Industrielle | Method and kit for the classification and prognosis of tissue or organ fibrosis in a reparative or reactive process |
KR20170031060A (ko) * | 2015-09-09 | 2017-03-20 | 중앙대학교 산학협력단 | Hapln1을 이용한 피부 노화 측정 또는 예방 또는 개선용 조성물 |
KR20200104831A (ko) * | 2019-02-27 | 2020-09-04 | 주식회사 하플사이언스 | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 |
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US20130052198A1 (en) | 2010-03-29 | 2013-02-28 | The General Hospital Corporation | Anti-inflammatory factors |
WO2013144349A1 (en) * | 2012-03-30 | 2013-10-03 | Societe De Developpement Et De Recherche Industrielle | Method and kit for the classification and prognosis of tissue or organ fibrosis in a reparative or reactive process |
KR20170031060A (ko) * | 2015-09-09 | 2017-03-20 | 중앙대학교 산학협력단 | Hapln1을 이용한 피부 노화 측정 또는 예방 또는 개선용 조성물 |
KR20200104831A (ko) * | 2019-02-27 | 2020-09-04 | 주식회사 하플사이언스 | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 |
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