KR100794391B1 - Pharmaceutical composition comprising comp-ang1 for unilateral ureteral obstruction - Google Patents

Abstract

A pharmaceutical composition comprising cartilage oligomeric matrix protein-angiopoietin-1 is provided to be used for treating the damage of endothelial cells and kidney fibrosis by protecting kidney endothelial cells. A pharmaceutical composition for treating unilateral ureteral obstruction comprises cartilage oligomeric matrix protein-angiopoietin-1. The composition is an oral formulation such as tablet, pill, powder, granule, syrup, liquid, suspension, emulsion and capsule or a non-oral formulation such as injection, transrectal formulation and transdermal formulation.

Description

Pharmaceutical composition for treating unilateral ureteral occlusion comprising cartilage oligomer matrix protein Angiopoin-1 (COOMP-Ang1) Pharmaceutical composition comprising COMP-Ang1 for Unilateral Ureteral Obstruction

Figure 1 shows protection of PECAM-1-positive endothelial cells by COMP-Ang1 in the kidney after unilateral ureteral occlusion.

Figure 2 shows the damage reduction of renal tubular epilepsy by COMP-Ang1 after unilateral ureteral occlusion.

Figure 3 shows the intercellular interstitial fibrogenesis, type I collagen precipitation, fibroblast activation and decreased expression of α-smooth muscle actin by COMP-Ang1 in unilateral ureteral occlusion kidney.

Figure 4 shows the increase in phosphorylation of Tie2 and Tie2 and Akt by COMP-Ang1 in unilateral ureteral occlusion kidney.

Figure 5 shows the effect of adenovirus COMP-Ang1 on the microcirculation of the kidney after unilateral ureteral occlusion.

FIG. 6 shows that COMP-Ang1 increases the peak of renal arterial blood velocity and renal cortex blood flow in unilateral ureteral occlusion mice.

FIG. 7 shows that COMP-Angl decreases the number of F4 / 80-positive cells infiltrated and modulates TGF-β1 and Smad in unilateral ureteral occlusion mice.

The present invention relates to a pharmaceutical composition for treating unilateral ureteral occlusion comprising cartilage oligomer matrix protein angiopotin-1 (COMP-Ang1). A pharmaceutical composition for treating unilateral ureteral occlusion that induces inhibition of fibrosis progression.

Most of chronic kidney disease progresses to tubular interstitial fibrosis. Deepening of interstitial changes is the most important measure of progression of kidney disorders (Risdon RA, Sloper JC, De Wardener HE: R Lancet 2: 363-366, 1968; Nath KA, Am J Kidney Dis 20: 1-17, 1992). Renal tubular interstitial fibrosis is a common feature in unilateral ureteral obstruction (UUO). Chronic and acute ureter blockages in humans can lead to various clinical conditions.

Damage to renal microvascular is a major contributing factor to renal disease progression (Bohle A, Mackensen-Haen S, Wehrmann M: Kidney Blood Press Res 19: 191-195, 1996). In particular, damage to the capillary network of the renal tubules is a major factor in tubular interstitial disease (Thomas SE et al., J Am Soc Nephrol 9: 231-242, 1998; Ohashi R, Kitamura H, J Am Soc Nephrol) 11: 47-56, 2000). Damaged angiogenesis can cause kidney disease and can damage the kidneys (Kang DH et al., J Am Soc Nephrol 12: 1434-1447, 2001).

Renal ischemia is a major contributing factor to the destruction of blood vessels and damage to the kidneys (Fine LG, Orphanides C, Norman JT, Kidney Int Suppl 65: S74-S78, 1998). Ohashi et al. (Ohashi R et al., J Am Soc Nephrol 13: 1795-1805, 2002) reported that degeneration of tubular capillaries contributes to wounds of tubular epilepsy in unilateral ureteral occlusion models.

 Therefore, endothelial cells (EC) play an important role in the progression of kidney disease in unilateral ureteral occlusion kidney. Renal microvascular injury is because growth factors or cytokines with endothelial protection or angiogenic efficacy in renal fibrosis contribute to an important mechanism that can improve renal fibrosis in unilateral ureteral occlusion models. (Yang J, Liu Y, J Am Soc Nephrol 13: 96-107, 2002; Morrissey J et al., J Am Soc Nephrol 13 [Suppl 1]: S14-S21, 2002; Chevalier RL et al., Kidney Int 57: 882-890, 2000).

Angiopoin-1 (Ang1) expresses ligands broadly against Tie2 tyrosine kinase receptors expressed in endothelial cells (Davis S et al., Cell 87: 1161-1169, 1996), regulating blood vessel growth, development, completion and permeability (Suri C et al., Cell 87: 1171-1180, 1996; Thurston G et al., Nat Med 6: 460-463, 2000). Angiopotin-1 is used as a potent therapeutic agent to induce angiogenesis, increase endothelial cell survival and prevent blood vessel leakage.

The inventors have found that angiopoin-1 increases endothelial cell survival and has an anti-inflammatory effect (Kim I et al., Circ Res 89: 477-479, 2001; Kwak HJ et al., Circulation 101: 2317-2324, 2000).

In addition, we have recently developed a cartilage oligomeric matrix protein (COM-Ang1), a variant of water-soluble and potent angiopoin-1 (Cho CH et al., Proc Natl Acad Sci USA 101: 5547). -5552, 2004). COMP-Ang1 is more potent than native Ang1 in phosphorylation of Tie2 and signaling by Akt in primary cultured EC.

COMP-Angl rescues apoptosis by radiation in microcapillary endothelial cells of intestinal villi (Cho CH et al., Proc Natl Acad Sci U S A 101: 5553-5558, 2004). In addition, the present inventors have recently demonstrated that continuous administration of COMP-Angl causes long-lasting vasodilation and increased blood flow (Cho CH et al., Circ Res 97: 86-94, 2005).

In kidney development Ang1 and Tie2 play an important role in the maturation of glomeruli and straight vessels (Yuan HT et al., J Am Soc Nephrol 10: 1722-1736, 1999). In addition, Ang1 expression is increased in folate-induced nephrotoxicity (Long DA et al., J Am Soc Nephrol 12: 2721-2731, 2001).

Meanwhile, the use of angiopoin-1 (Ang1) as a therapeutic agent in kidney disease has not been studied.

An object of the present invention is to provide a pharmaceutical composition for treating unilateral ureter occlusion comprising cartilage oligomeric matrix protein-angiopoietin-1.

The present invention for achieving the above object provides a pharmaceutical composition for treating unilateral ureter occlusion comprising cartilage oligomeric matrix protein-angiopoietin-1 (Cartilage oligomeric matrix protein-angiopoietin-1).

In the above, the pharmaceutical composition comprises an oral preparation comprising cartilage oligomer matrix protein Angiopotin-1.

In the above, oral preparations are characterized in that any one selected from the group consisting of tablets, pills, powders, granules, syrups, solutions, suspensions, emulsions and capsules.

In the above, the pharmaceutical composition comprises a parenteral preparation comprising cartilage oligomer matrix protein angiopotin-1.

In the above, the parenteral preparation is characterized in that any one selected from the group consisting of injectables, transectonic preparations and transdermal preparations.

Hereinafter, the content of the present invention will be described in more detail.

In the present invention, the pharmaceutical composition comprising cartilage oligomeric matrix protein-angiopoietin-1 can be effectively used for the treatment of unilateral ureteral obstruction.

The pharmaceutical composition of the present invention can be administered by oral or parenteral means. There is no particular limitation on the dosage form, but it will depend on the dosage form as well as the severity of the patient's age, sex or other condition and disease. Oral preparations can be used, for example, in the form of tablets, pills, powders, granules, syrups, solutions, suspensions, emulsions, or capsules. Examples of the parenteral preparation include injections, transrectal preparations, transdermal preparations, and the like, and can be administered by administration routes such as intravenous, subcutaneous muscle, and intraperitoneal.

In addition, the pharmaceutical composition of the present invention can be made into a desired dosage form by mixing with the pharmaceutically acceptable excipients, disintegrants, binders, lubricants, solubilizers, preservatives, stabilizers, buffers, coating agents and the like commonly used in the active ingredient.

Various carriers known in the art can be used for the tablet formulations above. Examples of typical carriers include: excipients such as lactose, saccharose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose and silicic acid; Binders such as water, ethanol, propanol, sweet syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate and polyvinyl pyrrolidone sugar and the like; Dry starch, sodium alginate, agar powder, naminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch and lactose Disintegrants such as; Disintegration aids such as saccharose, stearin, cacao butter and hydrogenated oils; Absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate; Wetting agents, such as glycerin and starch; Absorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; Lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol. If desired, tablets may be coated (eg, sugar coated, gelatin coated, enteric coated or film coated) and the tablets may be formed into bilayer or multilayer tablets.

For pill formulations, various carriers known in the art can be used. Examples include excipients such as glucose, lactose, starch, cacao oil, hydrogenated vegetable oils, kaolin and talc; Binders such as gum arabic powder, tragacanth powder, gelatin and ethanol; And disintegrants such as laminaran ager.

When formulated as an injection, sterile solutions or suspensions which are isotonic with blood are preferred. For liquid, emulsion or suspension formulations, any diluent commonly used in the art can be used. Examples include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, propoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In such cases, sodium chloride, glucose or glycerin may be included in an amount sufficient to prepare isotonic solutions or in amounts sufficient to add conventional dissolution aids, buffers, smoothing agents and the like. In addition, coloring agents, preservatives, aroma chemicals, flavoring agents, sweetening agents or other agents may be added if necessary.

Cartilage oligomer matrix protein Angiopotin-1 can be prepared as follows.

The recombinant protein for cartilage oligomer matrix protein angiopoietin-1 is a cartilage coiled oligomeric matrix that has a coiled coil domain present in the native native angiopoietin-1. can be prepared using recombinant CHO cell line (CA1-2; production rate, 30 mg / L) expressing COMP-Ang1 (refer to Cho CH et al. Proc Natl Acad Sci USA 2004; 101: 5547-5552 &2004; 101: 5553-5558).

The present invention provides whether cartilage oligomeric matrix protein angiopoietin-1 (COMP-Ang1) protects the endothelial cells of the kidney, has an anti-inflammatory effect or indirectly inhibits the progression of renal fibrosis. The unilateral ureteral occlusion was investigated.

According to a preferred embodiment of the present invention it can be seen that tubular damage and tubulointerstitial fibrosis occurring in unilateral ureteral occlusion mice are less than vehicle treated with COMP-Ang1 treatment.

The present invention can significantly inhibit the accumulation of α-smooth muscle actin-positive cells and interstitial type I collagen by COMP-Ang1 treatment. COMP-Angl protects kidney PECAM-1- and Tie2-positive endothelial cells and increases phosphorylation of Akt. After COMP-Angl treatment, renal surface microvessels are increased on in vivo vital microscopy and kidney blood flow is increased on laser Doppler ultrasonography compared to vehicle treatment.

COMP-Angl reduces mononuclear and macrophage infiltration, tissue concentrations of TGF-β1 and Smad 2 and 3 phosphorylation and increases Smad 7 in unilateral ureteral occlusion kidney.

These results of the present invention indicate that COMP-Angl treatment may inhibit the progression of renal fibrosis in ureteral obstruction.

The present invention shows that COMP-Angl treatment protects renal endothelial cells, reduces invasion of F4 / 80 positive cells, and reduces renal fibrosis in unilateral ureteral occlusion model. This indicates that COMP-Ang1 of the present invention can be used therapeutically for the treatment of endothelial cell damage and renal fibrosis.

According to the present invention, COMP-Ang1 protects extensively the renal failure by significantly improved morphology, interstitial fibrosis, and precipitation of collagen type I.

These results of the present invention suggest that COMP-Ang1 is a novel endothelial cell-specific therapeutic modality in fibrotic kidney disease.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention is not interpreted to be limited to these examples.

Example 1 Animal Experiment: Unilateral Ureter Obstruction (UUO) Model

Experimental Animals:

Tie2-GFP (green fluorescence protein) transgenic male mice (FVB / N; Jackson Laboratory, Bar Harbor, ME; 20-30 g body weight) were platelet-EC adhesion molecule-1 (PECAM-). 1) was used for immunostaining, and C57BL / 6 male mice (Charles River Korea, Seoul, Korea; 20-30 g body weight) were used for other experiments.

Unilateral Ureteral Occlusion (UUO) Models:

Male C57BL / 6 mice (Charles River Korea, Seoul, Korea; 20-30 g body weight) were cut in the midline of the abdomen and the exposed left adjacent urinary tract was clamped using 3-0 silk in two separate parts.

Preparation of Ade-COMP-Ang1 and Ade-LacZ (vehicle):

Recombinant protein for cartilage oligomer matrix protein angiopoietin-1 is a cartilage coil oligomeric matrix that has a coiled coil domain present in the native native angiopoietin-1. Substituted with matrix protein), and prepared using recombinant CHO cell line (Chinese hamster ovary cell) (CA1-2; production rate, 30mg / l) expressing COMP-Ang1. In addition, recombinant adenoviruses expressing COMP-Ang1 or LacZ through the pAdEasy vector system provided by Qbiogene were used to prepare Ade-COMP-Ang1 and Ade-LacZ. . Ade-sTie2-Fc was constructed as an adenoviral system by attaching the Fc domain to the Tie 2 extracellular domain (Ref. Cho CH, et al., Circ Res 97: 86-). 94, 2005).

Administration of Adenovirus:

For adenovirus treatment, Ade-COMP-Ang1, sTie2-Fc, and Ade-LacZ (vehicle) were each diluted in 50 μl of sterile 0.9% NaCl and administered intravenously through the tail vein.

In preliminary experiments, circulating serum concentrations of COMP-Ang1 increased after 3 days of administration and decreased after reaching peak on day 5.

To determine the effect of high COMP-Angl concentrations in unilateral ureter occlusion models, COMP-Angl adenovirus or vehicle adenovirus was administered to mice three days before and two weeks after unilateral ureter occlusion.

Example 2 Protection of Kidney PECAM-1- and Tie2-positive Endothelial Cells in Unilateral Ureteral Occlusion by COMP-Ang1

The serum concentration of COMP-Angl was 0 μg / ml on day 0, 2.8 ± 0.5 μg / ml on day 1, 3.9 ± 0.7 μg / ml on day 3, 4.3 ± 0.6 μg / ml on day 5, 3.5 ± on day 7 0.6 μg / ml, 1.7 ± 0.2 μg / ml at Day 14 and 0.2 ± 0.3 μg / ml at Day 28 (±: standard deviation; n = 5 in each group).

Since COMP-Ang1 is a potent angiogenic factor and ligand that expresses Tie2 in endothelial cells, it was examined whether COMP-Ang1 protects PECAM-1- and Tie2-positive endothelial cells of the kidney. Tie2-GFP transgenic mice were treated with COMP-Ang1 or vehicle.

Tie2-GFP (green fluorescence protein) introduced mouse kidney fragments administered with vehicle or COMP-Ang1 were stained with anti-PECAM-1 (red) (FIG. 1: n = 6; bar = 50 μm; Sham: pseudosurgical rats) ; CON: contralateral nasal obstruction kidney). After immunostaining, digital images were obtained through the same-focus microscope (Zeiss LSM 510 confocal microscope; Carl Zeiss, Germany), and the expression ratios of the unit areas were measured using a digital image analysis program (AnalySIS; Soft Imaging System).

After two weeks, there was a slight increase in PECAM-1-positive peritubule endothelial cells in unilateral ureteral occlusion kidney treated with vehicle compared to sham-operated kidney treated with vehicle, There was no difference in Tie2 in capillaries around the tubules of unilateral ureteral occlusion kidney treated with vehicle (FIG. 1).

COMP-Ang1 increases the expression of PECAM-1 and Tie2 in peritubule capillaries compared to unilateral ureteral occlusion kidney treated with vehicle (A in FIG. 1). After 4 weeks, expression of PECAM-1- and Tie2-positive endothelial cells decreased in unilateral ureteral occlusion kidney treated with vehicle compared to sham-operated kidney (FIG. 1B).

As shown in FIG. 1, expression of PECAM-1 positive endothelial cells and Tie2 in capillary capillaries was increased in unilateral ureteral obstruction kidney treated with COMP-Ang1 compared to vehicle-treated unilateral ureteral occlusion It was.

In vehicle treated mice, expression of PECAM-1 and Tie2 in the opposite kidney was unchanged at 2 and 4 weeks compared to sham-operated kidney.

In the contralateral and sham-operated kidneys, COMP-Ang1 slightly increased the expression of PECAM-1 and Tie2 at 2 and 4 weeks in COMP-Ang1 treated kidneys compared to vehicle-treated kidneys. I was.

In glomeruli, the expression of PECAM-1-positive endothelial cells and Tie2 was decreased in unilateral ureteral obstruction compared to sham-operated kidney, but COMP-Ang1 treated kidney compared to vehicle treated kidney. Increased in

The ratio of PECAM-1 expression area at 0.22 mm2 field area of the microscope is shown in Table 1 after 2 weeks and 4 weeks after vehicle administration in sham-operated kidney, contralateral kidney, and unilateral ureteral occlusion kidney, respectively. As shown.

TABLE 1 Ratio of PECAM-1 expression area in unilateral ureteral occlusion mice ^a (±: standard deviation, 15 microscope field)

Treatment (%) 2 wk after UUO 4 wk after UUO Sham CON UUO Sham CON UUO Vehicle 3.8 ± 0.2 3.6 ± 0.2 4.3 ± 0.2 ^b 3.3 ± 0.2 3.3 ± 0.3 0.5 ± 0.1 ^b COMP-Ang1 7.9 ± 0.2 ^c 7.8 ± 0.3 ^c 10.5 ± 0.9 ^{c, d} 7.6 ± 0.3 ^c 7.1 ± 0.2 ^c 0.2 ± 0.3 ^{c, d}

a COMP-Ang1: cartilage oligomer matrix protein- angiopotin-1; CON: opposite kidney; PECAM-1: platelet-endothelial cell adhesion molecule-1; Sham: pseudosurgical mouse; UUO: unilateral ureteral obstruction.

b P <0.05 compared to postoperative kidney on the same day in vehicle treated group.

c P <0.05 versus vehicle-treated height on the same day in each group.

d P <05 Psychosurgical kidney contrast on the same day in COMP-Ang1 treatment group.

Example 3 Morphological Protection of Tubular Epilepsy for Unilateral Ureteral Occlusion by COMP-Ang1

After unilateral ureteral occlusion, damage to renal tubular epilepsy by COMP-Ang1 was investigated (FIG. 2). Mice were administered 1 × 10 ⁹ pfu of adenovirus-COMP-Ang1 (Ade-COMP-Ang1). A and B indicate damage of tubular interstitial (A: low magnification; B: high magnification, PAS stain; bar = 50 μm). C is a semiquantitative calculation of tubular damage, with more damage in vehicle-treated kidneys than in COMP-Ang1-treated kidneys 2 and 4 weeks after unilateral ureteral occlusion (n = 8; ±: Standard deviation; gray bars: vehicle administration; red bars: COMP-Ang1 administration).

Two weeks after unilateral ureteral occlusion, occluded kidneys of mice administered vehicle showed destruction of the renal tubules with severe mononuclear inflammatory infiltration and mild interstitial fibrosis (FIGS. 2B and C). Unilateral ureteral occlusion kidney with COMP-Ang1 showed relatively well preserved renal tubule structure and less invasive inflammatory cells. In addition, interstitial fibrosis in the kidney administered COMP-Ang1 was lighter in the kidney administered vehicle (FIG. 2B).

After 4 weeks of unilateral ureteral occlusion, occlusion of the vehicle administered mice increased, the pelvis and calyces expanded, and a thin border of the remaining cortex was observed. In contrast, many renal soft tissues of unilateral ureteral occlusion kidneys in mice administered COMP-Ang1 were preserved (FIG. 2).

Histologically, unilateral ureteral occlusion elongation with vehicle showed more disruptive changes (FIG. 2). The tubules of the structurally preserved kidneys are not easily recognized and show severe interstitial fibrosis.

Infiltration of inflammatory cells in epilepsy decreased with the progression of fibrosis in the obstructed kidney. Unilateral ureter occlusion kidneys administered COMP-Ang1 were observed to better preserve the structure of mild fibrosis and renal tubules, similar to the administration of vehicle two weeks after unilateral ureter occlusion.

Sham-operated and contralateral kidneys showed no significant morphological changes after COMP-Ang1 administration compared to vehicle administered kidneys (FIGS. 2B and 2C).

Mice pre-exposed to COMP-Angl showed protection of 38% and 24%, respectively, 2 and 4 weeks after unilateral ureteral occlusion against histological damage of tubular epilepsy.

<Example 4> Improvement of renal fibrosis by unilateral ureteral occlusion with COMP-Ang1 treatment

To measure the effect of COMP-Angl on interstitial fibrosis, slides were stained with Masson's trichrome and collagen deposition was measured blue-stained.

Figure 3 shows the interstitial fiber formation of kidney, type I collagen precipitation, activation of fibroblasts and decreased expression of α-smooth muscle actin by COMP-Ang1 in unilateral ureteral occlusion kidney (n = 6; bar = 50 Μm: ±: standard deviation; gray bars: vehicle administration; red bars: COMP-Ang1 administration). A is a photomicrograph of Masson's trichrome stained tissue, and B is a semi-quantitative measure of fibrovascular interstitial fibrosis in Masson's trichrome stained tissue (n = 6). C shows immunochemical staining for collagen type I in unilateral ureteral occlusion kidney treated with pseudosurgical kidney, vehicle or COMP-Ang1, and D is the semi-quantitative value of type I collagen (n = 6). E is an immunochemical staining for α-smooth muscle actin in unilateral ureteral occlusion kidney treated with pseudosurgical kidney, vehicle or COMP-Ang1. arrow). F is the semiquantitative value of the α-smooth muscle actin-positive portion of tubular interstitial (n = 6). Hydroxyproline content at G was measured by colorimetric assay after acid hydrolysis and expressed according to the proportion of dry tissue weight (n = 6).

Collagen deposition increased in occlusion kidneys 2 and 4 weeks after unilateral ureteral occlusion (A, B in FIG. 3). Fibrosis was not observed in sham-operated and contralateral kidneys.

After 2 and 4 weeks of unilateral ureter occlusion, increased collagen deposition was 29% and 30%, respectively, in kidneys treated with COMP-Ang1 compared to vehicle-treated unilateral ureteral occlusion kidneys. Markedly decreased (B of FIG. 3). Type I collagen precipitated two and four weeks after surgery in unilateral ureteral obstruction kidney treated with vehicle compared to that in sham-operated kidneys (FIGS. 3C and 3). COMP-Angl significantly reduced the expression of collagen type I collagen in unilateral ureteral occlusion kidney by 64% and 37% after 2 and 4 weeks, respectively (FIG. 3D).

α-SMA is a marker of myofibroblast differentiation following occlusion. After 1 and 2 weeks, COMP-Ang1 reduced α-SMA expression by 37% and 31% compared to vehicle-treated unilateral ureteral occlusion kidney (E, F in FIG. 3).

Hydroxyproline content and total collagen content increased 3.5- and 6.5-fold after 2 and 4 weeks, respectively, in unilateral ureteral occlusion kidney treated with vehicle compared to that of pseudosurgical kidney. Ang1 decreased hydroxyproline content by 27% and 38%, respectively, due to unilateral ureter occlusion (G in FIG. 3). These results indicate that COMP-Angl improves renal fibrosis due to unilateral ureteral obstruction.

Example 5 Increase of Phosphorylation of Tie2 and Tie2 and Akt in Kidney in Unilateral Ureteral Infection by COMP-Ang1

The increase in phosphorylation of Tie2 and Tie2 and Akt by COMP-Ang1 was investigated in unilateral ureteral occlusion kidney (FIG. 4). A shows the immunodiffusion test of phosphorylation-Tie2, and the total protein of each lane was 150 µg, and was blotted with an anti-phospho-Tie2 antibody. Membranes were stripped and blotted with anti-actin antibodies to ensure equal protein levels in each lane. Densitometer analysis is shown according to the relative ratio of Tie2 to Actin and phosphorylation-Tie2 to Tie2. B shows the immune spot of phosphorylation-Tie2 when pre-treated with sTie2-Fc. Mouse, 1 × 10 ⁹ pfu process the Ade-COMP-Ang1 was treated with ^{1 × 10 9 pfu Ade-sTie2} -Fc for 24 hours. C represents an immunooblot of Akt, phosphorylated-Akt (phospho-Akt). The total kidney protein of each lane was 100 µg and blotted with an anti-phospho-Akt antibody. Membranes were stripped and blotted with anti-Akt antibodies to ensure the same protein levels in each lane. Densitometer analysis was performed according to the relative ratio of phosphorylation-Akt2 to Akt.

COMP-Angl acts on endothelial cell receptor Tie2 to phosphorylate Tie. In Western blot analysis, COMP-Ang1 increased phosphorylation of Tie2 and Tie2 5, 7 and 14 days after unilateral ureteral occlusion compared to vehicle (A in FIG. 4). At 5 days after COMP-Ang1 treatment, phosphorylation of Tie2 and Tie2 increased 1.3-fold and 1.25-fold, respectively, indicating that the treatment of COMP-Ang1 increased the phosphorylation of Tie2 and Tie2 of endothelial cells.

To determine the relationship between Tie2 in COMP-Ang1-induced phosphorylation of Tie2 in unilateral ureteral occlusion kidney, we pretreated mice with sTie2-Fc 24 hours before COMP-Ang1 treatment. The pretreatment was contrary to the phosphorylation of COMP-Ang1-induced Tie2 5 days after surgery (FIG. 4B), indicating that the phosphorylation of COMP-Ang1-induced Tie2 occurs through activation of Tie2 in the kidney.

Since phosphorylation of Akt in endothelial cells is an important sub-path of COMP-Angl, we analyzed phosphorylation of Akt in unilateral ureteral occlusion kidney treated with COMP-Angl. As a result, COMP-Ang1 increased the phosphorylation of Akt at 5 days, 7 days and 14 days after unilateral ureteral obstruction compared to vehicle (FIG. 4C). Phosphorylation of Akt increased 1.7, 1.4 and 1.3 times at 5, 7 and 14 days, respectively.

Example 6 Protection of Renal Microvascularity in In Vivo Vital Microscopy of COMP-Ang1

The effect of adenovirus COMP-Angl on the microcirculation of the kidney after unilateral ureteral occlusion was investigated (FIG. 5). A represents the microcirculation of the kidney after COMP-Ang1 or vehicle administration (bar = 100 μm). E in B represents a three-dimensional evaluation of the characteristics of the microvascular. Individual frame images of the microvasculature surface of the kidney after COMP-Ang1 or vehicle administration were analyzed (Cascade 650 CCD camera, Roper Scientific, Tucson, AZ; MetaMorph software, Universal Imaging Corp). , Sunnyvale, CA). B is the volume fraction of blood vessels in unilateral ureteral occlusion kidney, C is the functional microvascular concentration, D is the branch-point density, and E is the vessel segment length.

A decrease in the surface area of microvessels was observed after COMP-Ang1 or vehicle treatment in unilateral ureteral obstruction kidney compared to sham-operated kidney, but vehicle-treated unilateral ureter occlusion kidney In some residual vessels abnormally expanded and bent.

COMP-Ang1 reduced the reduction of microvascular surface area due to unilateral ureter occlusion and improved abnormal vascular swelling and bend compared to unilateral ureter occlusion kidney treated with vehicle (FIG. 5A).

According to the map of the functional microvascular vessel using A of FIG. 5, the volume fraction of the blood vessel, functional after 2 weeks and 4 weeks in unilateral ureteral occlusion kidney treated with vehicle compared to sham-operated kidney Microvascular concentration and branch-point density were reduced (B, D in FIG. 5). After 2 weeks, 41%, 39%, 57% decreased, and after 4 weeks, 54%, 48%, 65% decreased, respectively.

As branch-point density decreased in vehicle-treated unilateral ureteral occlusion kidneys, vascular fragment lengths increased by 180% and 225%, respectively, compared to sham-operated kidneys (Fig. 5, E).

However, in unilateral ureteral obstruction kidney treated with COMP-Ang1, unilateral ureteral occlusion kidney treated with vehicle at 2 and 4 weeks of volume fraction, functional microvascular concentration and branch-point density of blood vessels It was significantly higher than in (Fig. 5 B, D). Two weeks later, 140%, 135%, and 184% increased, and after four weeks, 153%, 132%, and 185%, respectively.

COMP-Ang1 reduced fragment length by 23% and 16% at 2 and 4 weeks, respectively, compared to vehicle-treated unilateral ureteral occlusion kidney (FIG. 5E).

These results indicate that COMP-Ang1 reduces vascular volume, length and branch-point density due to unilateral ureter occlusion and decreases due to unilateral ureter occlusion at vessel segment length. .

Example 7 Increased Velocity of Renal Cortex and Renal Artery by COMP-Ang1 in Unilateral Ureteral Occlusion Mice

In unilateral ureteral occlusion mice, the peak of renal arterial blood velocity and increased blood flow in renal cortex were investigated by COMP-Angl (FIG. 6). A is the mean systemic arterial BP, B is the blood flow of the renal cortex, C is the highest renal arterial blood rate, and the anesthesia was measured by anesthesia (n = 6, gray bar: vehicle treatment; red bar: COMP). -Processing Ang1).

The effect of COMP-Ang1 on blood pressure (BP), renal cortex and maximum velocity of renal artery in systemic artery in unilateral ureteral occlusion mice is shown in FIG. 6.

Compared with sham-operated mice, after vehicle or COMP-Ang1 treatment, the systemic mean arterial pressure of unilateral ureteric occlusion mice was not significantly different (FIG. 6A), but the neocortex Blood flow decreased significantly to 65% and 92% at 2 and 4 weeks, respectively, in vehicle treatment (B of FIG. 6).

After COMP-Ang1 treatment, blood flow in renal cortex was significantly increased by 1.6 and 2.9 times at 2 and 4 weeks, respectively, compared to vehicle treatment (B of FIG. 6). Two weeks after unilateral ureteral occlusion, the maximum rate of renal artery did not change significantly compared to sham-operated mice.

Although the maximum rate of renal artery in the kidney treated with COMP-Ang1 increased 1.2 times in 2 weeks compared to sham-operated mice, the maximum of renal artery at 2 weeks compared to vehicle treated mice. There was no significant difference in speed.

However, compared to sham-operated mice, the maximum rate of renal artery decreased by 30% at week 42 when vehicle was treated, and after COMP-Ang1 treatment compared to vehicle treated mice. At week 42, the maximum rate of renal artery increased 1.7-fold (FIG. 6C).

These results indicate that COMP-Angl increases the maximal rate of blood flow and renal artery of the renal cortex in unilateral ureteral occlusion mice.

Example 8 Reduction of F4 / 80-positive Cell Infiltration After COMP-Ang1 Treatment

We examined the infiltration of F4 / 80-positive cells and the regulation of TGF-β1 and Smad by COMP-Ang1 in unilateral ureteral occlusion mice (FIG. 7). A is a micrograph of F4 / 80 staining (bar = 50 μm), B is the number of infiltrated F4 / 80-positive cells (n = 6; gray bar: vehicle treatment; red bar: COMP-Ang1 treatment), C is Mice show the number of infiltrated F4 / 80-positive cells (n = 5) after pretreatment with 1 × 10 ⁹ pfu Ade-sTie2-Fc 24 hours prior to treatment with 1 × 10 ⁹ pfu Ade-COMP-Ang1.

The number of monocytes and macrophages was determined by immunohistochemistry of the F4 / 80 antigen per unit area of the kidney treated vehicle, according to sham- 7 times and 10 times increased after 1 week and 2 weeks after unilateral ureteral obstruction compared to kidneys (A, B of FIG. 7). COMP-Ang1 significantly reduced the number of F4 / 80-positive cells by 31% and 45%, respectively, at 1 and 2 weeks compared to unilateral ureteral occlusion kidney administered vehicle (FIG. 7). A, B).

Pretreatment of sTie2-Fc was contrary to the effect of COMP-Angl on infiltration of F4 / 80-positive cells (FIG. 7C). Thus, treatment with COMP-Angl reduced monocyte and macrophage infiltration via Tie2 in unilateral ureteral occlusion kidney.

Example 9 Changes of TGF-β1, Smad 2 and 3, and Smad 7 in Kidney after COMP-Ang1 Treatment

TGF-β1 plays a central role in the interstitium macrophage number and renal fibrosis in human clinical cases as well as in animal studies and is highly involved in TGF-β1 mRNA levels in the neocortex.

At 5, 7 and 14 days after surgery, the concentration of activated TGF-β1 in the tissues of the surgeon or unilateral ureteral occlusion kidney was measured by enzyme-linked immunoassay (ELISA) (FIG. 7D). E is phosphorylated-Smad 2/3 F shows the result of immuno-spot analysis of Smad 7. Blots were phosphorylated-Smad 2/3 or Smad 7 antibodies as searchers. Membranes were stripped and instilled with anti-actin antibodies to ensure equal protein levels in each lane. Densitometer analysis is shown according to the relative ratio of phospho-Smad 2/3 or Smad 7 to actin.

Quantitation by specific enzyme immunoassay (ELISA) showed a markedly increased tissue concentration in unilateral ureteral occlusion kidneys treated with a vehicle of active TGF-β1 (FIG. 7D). After COMP-Ang1 treatment, kidney TGF-β1 decreased 48%, 31%, and 35% at 5, 7, and 14 days of unilateral ureteral obstruction, respectively, compared to vehicle treatment.

Subregulation of Smad 3 is associated with attenuated renal fibrosis in unilateral ureteral occlusion models, and Smad 7 negatively regulates renal fibrogenesis by inhibiting Smad 2 and 3 activity in vivo.

Immunoblot increased 4.1-fold and 2.9-fold phospho-Smad 2 and 3 in kidneys after 14 days in unilateral ureteral occlusion kidney with vehicle compared to sham-operated mice. (E of FIG. 7). After COMP-Ang1 administration, phospho-Smad 2 and 3 decreased 29% and 41%, respectively, after 14 days in unilateral ureteral occlusion kidney compared to vehicle administration (E in FIG. 7).

After 14 days, Smad 2 was reduced by 42% in unilateral ureteral obstruction kidney administered vehicle (vehicle) compared to sham-operated mice; Administration of COMP-Angl increased Smad 7 by 1.2-fold compared to vehicle administration (FIG. 7E).

These results suggest that COMP-Angl lowers the increase in Smad 2 and 3 phosphorylation and decrease in Smad 7 induced by unilateral ureteral occlusion.

As described above, the cartilage oligomer matrix protein angiopotin-1 of the present invention may be used for the treatment of endothelial cell damage and renal fibrosis by protecting renal endothelial cells in unilateral ureteral occlusion.

Claims (5)

A pharmaceutical composition for treating unilateral ureteral occlusion comprising cartilage oligomeric matrix protein-angiopoietin-1. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an oral formulation comprising cartilage oligomer matrix protein Angiopotin-1. The pharmaceutical composition according to claim 2, wherein the oral preparation is any one selected from the group consisting of tablets, pills, powders, granules, syrups, solutions, suspensions, emulsions and capsules. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a parenteral preparation comprising cartilage oligomer matrix protein Angiopoin-1. The pharmaceutical composition according to claim 4, wherein the parenteral preparation is any one selected from the group consisting of injections, transrectal preparations and transdermal preparations.

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