Fusion protein Slit2D2(C386S)-HSA and use thereof in treatment of fibrotic diseases
10947296 ยท 2021-03-16
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
C07K2319/31
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the field of biomedical technology, in particular to a fusion protein Slit2D2(C386S)-HSA and use thereof in the treatment and/or prevention of fibrotic diseases. In the fusion protein, the amino acid residue is mutated on the basis of the Slit2D2 domain, which improves the stability of the fusion protein compared with the native protein. The above fusion protein is obtained by fusing Slit2D2(C386S) with HSA protein, which prolongs the metabolism time of the drug while improving the stability of the drug. The fusion protein provided by the present invention is more effective than the positive control drug in the prevention and treatment of fibrotic diseases, particularly pulmonary fibrosis, and shows good drug-forming properties.
Claims
1. A polypeptide or protein comprising or consisting of a D2 domain of a Slit2 protein, the Slit2 protein is derived from human, wherein the cysteine in the D2 domain of the Slit2 protein, corresponding to the 5.sup.th cysteine of the Slit2 protein, is located at position 386 of the Slit2 protein and is mutated to a polar amino acid.
2. The polypeptide or protein of claim 1, wherein the polar amino acid is selected from the group consisting of amino acid residues of Ser, Gln, Thr, Asn and Tyr.
3. The polypeptide or protein of claim 2, wherein the polypeptide or protein comprises or consists of the amino acid sequence as shown in SEQ ID NO: 1.
4. A nucleotide encoding the polypeptide or protein of claim 1.
5. The encoding nucleotide of claim 4 having the nucleotide sequence set forth in SEQ ID NO: 2.
6. A fusion protein comprising the polypeptide or protein of claim 1.
7. The fusion protein of claim 6, wherein the polar amino acid is selected from the group consisting of amino acid residues of Ser, Gln, Thr, Asn and Tyr.
8. The fusion protein of claim 6, wherein the polypeptide or protein comprises or consists of the amino acid sequence as shown in SEQ ID NO: 1.
9. The fusion protein of claim 6, wherein the fusion protein further comprises human serum albumin.
10. The fusion protein of claim 9, wherein the fusion protein comprises or consists of the amino acid sequence as shown in SEQ ID NO: 3.
11. A nucleotide encoding the fusion protein of claim 6.
12. The nucleotide of claim 11, wherein the nucleotide comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 4.
13. A method for preventing and/or treating a fibrotic disease or sepsis comprising a step of administering a pharmaceutical composition containing an effective amount of the fusion protein of claim 6.
14. The method of claim 13, wherein the polypeptide or protein comprises or consists of the amino acid sequence as shown in SEQ ID NO: 1.
15. The method of claim 14, wherein the fusion protein further comprises human serum albumin.
16. The method of claim 13, wherein the fusion protein comprises or consists of the amino acid sequence as shown in SEQ ID NO: 3.
17. The method of claim 16, wherein a nucleotide encoding the fusion protein comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 4.
18. The method of claim 13, wherein the fibrotic disease is pulmonary fibrosis, and/or, the sepsis is severe sepsis or septic shock.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(21) The technical solutions in the embodiments of the present invention will be clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
Example 1
Preparation of the Fusion Protein Slit2D2(C386S)-HSA
(22) Based on the known sequence of Slit2 [GenBank: EAW92793.1], the second domain of Slit2, Slit2D2, was analyzed, designed and constructed, and Slit2D2(C386S) was designed as shown in SEQ ID NO: 1, and further, the sequences encoding Slit2D2(C386S) and Slit2D2(C386S)-HSA were designed as shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
(23) The encoding sequence of Slit2D2(C386S)-HSA was obtained by total gene synthesis, and inserted into pCDNA3.4 (Brand: Thermo, Art. No.: A14697) expression vector by T/A clone. The map of the recombinant vector pCDNA3.4-Slit2D2(C386S)-HSA is shown in
(24) The recombinant plasmid in E. coli TOP10 was extracted with an endotoxin-free plasmid extraction kit for transfection into ExpiCHO-S cells (Gibco Catalog No. A29127). ExpiCHO-S cells were cultured and transfected with the recombinant plasmid when the cell density reached 410.sup.6-610.sup.6 cells/ml (transfection reagent: ExpiFectamine CHO Transfection Kit, Gibco Catalog No. A29129). After transfection, the cells were cultured for 10 days. The supernatant was collected, centrifuged at high speed, and purified through a HSA affinity chromatography (Filler of chromatography: Thermo, Art. No.: 191297050) and a weak anion exchange chromatography (Brand: Smart-Lifesciences, Art. No.: DEAE Beads 6FF, SI005025) to give the Slit2D2(C386S)-HSA fusion protein.
(25) The molecular weight of the purified fusion protein was determined by SDS-PAGE method. The purity of the fusion protein was determined by SEC-HPLC. The results of SDS-PAGE and the sepctra of SEC-HPLC are shown in
Example 2
Determination of the Affinity of the Fusion Protein to Target Robo1 Protein by SPR
(26) The affinity constant between the protein and Robo1 protein was detected by SPR (Surface Plasmon resonance BIAcore 200) method. The Robo1 protein (ORIGEN, Art. No.: TP327713) was bound to a CM5 chip, and the interaction between the fusion protein Slit2D2(C386S)-HSA (prepared in Example 1), Slit2D2-HSA (as disclosed in Patent Application PCT/CN2015/092079), and the receptor protein Robo1 were analyzed. Kinetic measurements were performed according to the method referred by Canziani et al. (2004, Anal. Biochem. 325: 301-307). Furthermore, the affinity of the Slit2N protein (Slit2N is a protein having a molecular weight of about 120 kDa at the N-terminus of the Slit2 protein) to Robo1 protein was also determined by the same method. The results are shown in Table 1.
(27) TABLE-US-00001 TABLE 1 Results of the affinity of the fusion protein to receptor Robo1 determined by SPR No. Protein Ka(1/Ms) KD(M) Kd(nM) 1 Slit2N 7.436 10.sup.5 3.394 10.sup.1 4.5 2 Slit2D2-HSA 1.771 10.sup.5 5.099 10.sup.4 2.8 3 Slit2D2(C386S)-HSA 8.647 10.sup.5 1.167 10.sup.1 1.35
(28) The results show that both of the designed and constructed fusion proteins Slit2D2-HSA and Slit2D2(C386S)-HAS have good affinity to the receptor Robo1 protein, which have similar properties to the Slit2N protein.
Example 3
Determination of the Protein Stability by ELISA
1. Reagents
(29) Neutroavidin-HRP diluent;
(30) Coating buffer: 0.16% Na.sub.2CO.sub.3; 0.3% NaHCO.sub.3; pH 9.8;
(31) Washing buffer: PBS containing0.1% Tween20;
(32) Blocking buffer: Washing buffer containing1% Goat Serum;
(33) TMB: purchased from Shanghai Beyotime Biotechnology Co., Ltd.;
(34) Stop solution: purchased from Shanghai Beyotime Biotechnology Co., Ltd.;
(35) Note: All antibodies were diluted with the blocking buffer.
2. Experimental Process
(36) The Robo1 protein was diluted to 1 g/ml, and was used to coat a plate at 100 l/well overnight at 4 C. The plate was washed 3 times with Washing buffer, and blocked with Blocking buffer (200 l/well) for 2 hours at room temperature, then washed 3 times with Washing buffer. The test samples (fusion protein Slit2D2(C386S)-HSA (prepared in Example 1), Slit2D2-HSA (as disclosed in Patent application PCT/CN2015/092079) 100 l) were separately added and incubated at room temperature for 2 hours. The plate was washed 3 times with Washing buffer. Anti-HSA-HRP antibody was diluted to 1 g/ml at 1:10000, and was used to coat the plate at 100 l/well, and then incubated for 1 hour at room temperature. The plate was washed 3 times with Washing buffer. 100 l of TMB was added to each well to develop color for 15 minutes. Color development was stopped by adding 50 l of Stop solution to each well. The absorbance at 450 nm was read, and the experimental results are shown in
(37) The results show that the EC.sub.50 values of Slit2D2(C386S)-HSA and Slit2D2-HSA were 10.56 and 214.6 nM, respectively, indicating that Slit2D2(C386S)-HSA has better stability than Slit2D2-HSA protein after treatment.
Example 4
Pharmacokinetic Assay
1. Experimental Animals
1.1 Basic Information
(38) Lines and sources: cynomolgus monkeys, Guangxi Xiongsen Primate Laboratory Animal Breeding Development Co., Ltd.
(39) Animal Stock Centre: 999M-014, non-nave;
(40) Age of experimental animals: 3.0-4.5 years old;
(41) Body weight of animals before the start of the test: 2.75-3.00 kg;
(42) Number and gender: 2 males, and 2 females.
1.2 Animal Feeding
(43) Each animal was housed in a single cage (stainless steel mobile cage) in an environmentally controlled room in the test facility. The temperature and relative humidity of the room were recorded twice a day. The temperature and relative humidity during the test were in the range of 18 to 26 C. and 40 to 70%, respectively. The actual temperature and relative humidity records were saved in the original record. The animals are illuminated, alternating between light and dark, for about 12 hours each day.
(44) The compound feed (Production batch numbers: 1650230222 and 1650230527, expiry dates: 2016 May 21 and 2016 Aug. 26, respectively, purchased from Beijing Huafukang Bioscience Co., Inc.) was freely fed by the experimental monkeys during the test. The compound feed for the experimental monkeys was tested by a third party (PONY) commissioned by the test facility to determine specific microorganisms, heavy metals and pesticide residues in the feed. The reverse osmosis water was supplied to each animal through a water bottle without interruption. The pH, hardness, heavy metals and microorganisms of the drinking water were regularly tested by the applicant and the commissioned third party. The test results of feed and water were in compliance with the relevant national regulations.
2. Main Computer Software or Computer System Used in the Test
(45) TABLE-US-00002 TABLE 2 Main computer software or computer system used in the test Medicilon Pharmaceutical Technology (Shanghai) Co., Ltd. Microsoft Office 2003 Professional Edition A set of utilities for working Microsoft Office 2007 Professional Edition: with text and data. Including 3. Test method Word and Excel, etc. 3.1 Test design Phoenix Pharmacokinetic Software
(46) TABLE-US-00003 TABLE 3 Test design table Concentration Body Dosage of of Volume of Amount of Route Animal weigth administration administration administration administration of No. Group Gender Test substance (kg) (mg/kg) (mg/mL) (mL/kg) (mL) administration* 101 1 Male Slit2D2(C386S)-HS 2.70 2.0 1.0 2.0 5.4 IV A 102 1 Male Slit2D2(C386S)-HS 2.65 2.0 1.0 2.0 5.4 IV A 103 1 Female Slit2D2(C386S)-HS 2.75 2.0 1.0 2.0 5.6 IV A 104 1 Female Slit2D2(C386S)-HS 2.60 2.0 1.0 2.0 5.2 IV A *Single administration.
3.2 Administration
3.2.1 Mode of Administration
(47) The test substance was administered in a single dose by intravenous injection.
3.3 Detection Indicators
3.3.1 Observation
(48) During the test, all animals were observed at various time points in the blood collection and before administration, including morbidity, damage, death rate and food and water supply.
3.4 Pharmacokinetics and Immunogenicity Studies
3.4.1 Number of Animals
(49) All animals did not need to be fasted before sampling.
3.4.2 Biological Sample Collection
(50) Blood was collected through the eyelids, and about 2.0 mL of each sample was collected and placed in an Eppendorf tube containing 200 L of 3.8% sodium citrate, and then placed on ice.
3.4.2.1 Sampling Time
(51) Sampling was performed once before administration (0 h), and at 3 h, 6 h, 12 h, 24 h and 4th, 6th, 8th, 11th, 14th, 17th, 19th, 22nd, 26th, 36th, and 43th day after administration, with a total of 17 time points.
3.4.3 Plasma Sample Processing
(52) The blood samples were collected and centrifuged to separate plasma (centrifugation conditions: 8000 rpm, 6 minutes, 2-8 C.). The contents of the label included: the subject number of Medicilon Pharmaceutical Technology (Shanghai) Co., Ltd., relevant test days, serial number of animals, date, and sampling time point. The collected plasma samples were stored in a refrigerator at 65 C. before analysis. After analysis, the remaining plasma samples were stored in the refrigerator at 65 C. for subsequent processing.
3.4.4 Sample Analysis
(53) The biological sample analysis method and analysis of all samples were performed by the analytical laboratory of Medicilon Pharmaceutical Technology (Shanghai) Co., Ltd. Each plate in the sample analysis must contain a standard curve. At least of the points and not less than 6 non-zero concentration points (without anchor points) constituting the standard curve should meet the acceptance criteria for method validation. Each plate should also contain at least 2 sets and each set comprised at least 3 quality control samples of different concentration levels (high-concentration quality control, intermediate-concentration quality control, and low-concentration quality control). It was further required that the quality control sample, which accounts for 67% () of the total and has each concentration level not lower than 50%, should have a precision within 20% and an accuracy of 80-120%.
3.5 Disposal of the Animals
(54) Blank plasma was collected from all the experimental animals at the end of the test and transferred to the animal stock centre of the test facility. The disposal of the animal was recorded.
4. Test Results
4.1 Observation
(55) No abnormal findings were observed during the test.
4.2 Determination of Plasma Drug Concentration
(56) The determination results of plasma drug concentration of cynomolgus monkeys after intravenous injection of Slit2D2(C386S)-HSA are shown in Table 4. The drug concentration-time curves are shown in
(57) Plasma drug concentration-time trends in animals of different genders (except #101-Male) in the Slit2D2(C386S)-HSA group are basically consistent or vary to some extent.
(58) TABLE-US-00004 TABLE 4 Plasma drug concentration of cynomolgus monkeys after a single intravenous administration of 2 mg/kg of Slit2D2(C386S)-HSA Slit2D2(C386S)-HSA-IV-2 mg/kg Plasma drug concentration (ng/mL) Time point (day) 101-Male 102-Male 103-Female 104-Female Mean SD 0 0.00 0.00 0.00 0.00 NA NA 0.125 16151.832 12629.116 13162.76 13541.873 13871.40 1565.72 0.25 15501.583 13612.467 11266.684 12289.107 13167.46 1828.53 0.5 13047.082 13169.983 9508.594 10597.55 11580.80 1819.92 2 11326.279 8055.924 5952.269 8296.324 8407.70 2212.34 5 6773.314 4063.179 3914.939 3663.777 4603.80 1455.71 6 2717.981 2215.633 2155.966 1433.272 2130.71 528.88 8 1577.577 BLQ 731.688 BLQ 1154.63 NA 11 BLQ BLQ BLQ BLQ NA NA 14 BLQ BLQ BLQ BLQ NA NA 17 BLQ BLQ BLQ BLQ NA NA 19 BLQ BLQ BLQ BLQ NA NA 22 BLQ BLQ BLQ BLQ NA NA 26 BLQ BLQ BLQ BLQ NA NA 29 BLQ BLQ BLQ BLQ NA NA 36 BLQ BLQ BLQ BLQ NA NA 43 BLQ BLQ BLQ BLQ NA NA NA: None/Not applicable; BLQ: Below the minimum limit of quantitation; LLOQ = 1 ng/mL
4.3 Pharmacokinetic Parameters
(59) The pharmacokinetic parameters were calculated with non-compartmental model using Phoenix pharmacokinetic software: AUC.sub.0-t, AUC.sub.0-, C.sub.max, t.sub.1/2, T.sub.max, C0, Cl, MRT.sub.0- and Vss.
(60) The pharmacokinetic parameters of Slit2D2(C386S)-HSA administered intravenously are shown in Table 5.
(61) The ratio of t.sub.1/2 between male and female in Slit2D2(C386S)-HSA group was 1.24, the ratio of C.sub.max was 1.11, and the ratio of AUC(.sub.0-) was 1.41. The main pharmacokinetic parameters (t.sub.1/2, C.sub.max and AUC(.sub.0-)) of animals of different genders in the group are basically consistent or vary to some extent, and the ratio difference ranges from 1.11 to 1.41.
(62) TABLE-US-00005 TABLE 5 Some pharmacokinetic parameters of cynomolgus monkeys after a single intravenous administration of 2 mg/kg of Slit2D2(C386S)-HSA Animal No. t.sub.1/2 T.sub.max C.sub.max C0 AUC.sub.(0-t) AUC.sub.(0-) CL MRT.sub.(0-) Vss Gender day day ng/mL ng/mL ng/mL*day ng/mL*day mL/day/kg day mL/kg 101-Male 2.04 0.13 16151.83 16829.36 45588.03 50239.09 39.81 2.84 113.09 102-Male 2.15 0.25 13612.47 12629.12 30270.94 37136.49 53.86 2.81 151.45 103-Female 1.82 0.13 13162.76 15377.93 28598.53 30524.24 65.52 2.58 168.88 104-Female 1.56 0.13 13541.87 14922.35 28034.90 31263.33 63.97 2.10 134.46 Mean 1.89 0.16 14117.23 14939.69 33123.10 37290.79 55.79 2.58 141.97 SD 0.26 0.06 1370.70 1741.83 8364.03 9125.03 11.84 0.34 23.84
Example 5
Evaluation of Efficacy on Pulmonary Fibrosis Model
1. Experimental Materials
1.1 Experimental Animals
(63) Experimental animals: SD rats, SPF grade, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., animal certificate number: 11400700171426.
1.2 Molding Agent
(64) Boremycin hydrochloride for injection, purchased from Nippon Kayaku Co., Ltd.;
(65) Specification: 15 mg/bottle;
(66) Batch number: Y50512;
(67) Production date: Jun. 8, 2015;
(68) Period of validity: until Jun. 7, 2017.
1.3 Solvent
(69) Normal saline: Anhui Double-Crane Pharmaceutical Co., Ltd., product batch number: 160502 8T;
(70) Methyl cellulose: Sigma, Art. No.: M0512-100G, product batch number: 079K0054V;
(71) Tween 80: Aladdin, Art. No.: T104865-500 ml, product batch number: K1519036;
(72) DMSO: NA, Art. No.: NA, product batch number: LE20Q62;
(73) PEG400: Sigma, Art. No.: NA, product batch number: MKBG7718V.
1.4 Preparation of Test and Control Products
(74) Preparation of 0.5% MC/0.2% Tween 80 vehicle: 100 mL of DDW was heated to 80-90 C., and 5 g of methyl cellulose was added thereto and stirred well. The heat source was removed, and about 400 mL of ice DDW was added thereto. The mixture was stirred for 30 minutes in an ice bath, and then the solution was transferred to a 1 L volumetric flask. After returning to room temperature, DDW was added thereto to a final volume of 1 L, and stirred until a clear solution was obtained. 2 ml of Tween 80 was added to 1 L of 0.5% methylcellulose solution, dissolved, and vortexed to obtain a homogeneous solution, which was stored at 4 C. until use.
(75) Preparation of 5.0 mg/ml PFD solution: 1440 mg of PFD powder was weighed and placed in a brown dispensing bottle, and 288 mL of 0.5% MC/0.2% Tween 80 solution was added thereto, and then the resulting mixture was subjected to an ultrasonic water bath to obtain a homogeneous solution, which was allowed to stand for 3 days at 4 C. and then reformulated.
(76) Slit2D2 (C386S)-HSA was diluted with PBS.
(77) Positive control: Pirfenidone, abbreviated as PFD, diluted with PBS.
2. Experimental Methods
2.1 Animal Feeding
(78) Male SD rats, 24, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals are kept in a SPF barrier system of an Animal Centre of Nanjing Baijiahui Medicine Research and Development Platform, which followed the international standard temperature, humidity and light control system.
2.2 Model Establishment
(79) Animals were anesthetized by inhaling isoflurane. After confirmation of anesthesia, the animals were sterilized, then the neck skin was cut, the muscles were bluntly separated to expose the trachea, and bleomycin (dosage: 3 mg/kg, volume: 1.0 mL/kg) was directly injected between the tracheal rings. After the operation, the animals were placed in a 37 C. electric blanket to keep warm until the animals were completely awakened. After confirming that they were able to eat and drink freely, the animals were returned to the cage for normal feeding.
2.3 Experimental Grouping
(80) In this experiment, there were four groups, i.e., model group (experimental group-1, n=60), PFD group (experimental group-2, n=6), Slit2D2(C386S)-HSA-1 mg/kg (experimental group-3, n=6), and Slit2D2(C386S)-HSA-5 mg/kg (experimental group-4, n=6). The specific information of the experimental grouping is shown in Table 6.
(81) TABLE-US-00006 TABLE 6 Experimental grouping Number Ad- of Compound minis- Left lung Grouping animals Modeling therapy tration pathology Experimental 6 Vehicle PO 6 group-1 QD. Experimental 6 PFD, 50 mg/kg PO 6 group-2 BID. Experimental 6 Slit2D2(C386S)- Iv 6 group-3 HSA, Q2D. 1 mg/kg Experimental 6 Slit2D2(C386S)- Iv 6 group-4 HSA, Q2D. 5 mg/kg
2.4 Test for Administration
(82) In this experiment, the positive control drug (PFD) was given to rats by gavage twice a day, which was started on the day of modeling, and administered continuously for 14 days. The test compound (Slit2D2(C386S)-HSA) was given intravenously once every other day, which was started on the day of modeling, and a total of seven doses were administered (as shown in table 6).
2.5 Physiological Observation of the Experimental Animals
(83) Physiological observation of the experimental animals: changes in body weight of the animals were measured (body weight was measured once a day before administration); and the death rate of animals during the test period was monitored.
2.6 Test Endpoint
(84) Animals were euthanized 24 hours after the last administration on the 14th day of modeling. After confirming the death of the animals, the left lung was fixed by intrapulmonary infusion of formalin, and the volume and weight of the left lung after perfusion were measured for relevant examination of the pulmonary pathology.
2.7 Pulmonary Pathological Examination
(85) General pathological examination: After the perfusion with an equal amount of formalin in the left lung, the wet weight of left lung after perfusion was weighed/recorded with a micro-balance; and the volume of left lung after perfusion was measured and recorded using a micro-measuring cup.
(86) Pathological examination of lung tissue: The whole lung was dehydrated. Lung paraffin sections were made with paraffin blocks. The HE-stained sections had a thickness of 3 m, and the Masson Trichrome-stained sections had a thickness of 4 m. HE staining and Masson Trichrome staining were performed according to pathological staining SOP, and whole section scanning was performed using a Digital Pathscope slice scanner. Pathological analysis and scoring of changes in damage and inflammation of terminal bronchioles and accompanying small pulmonary artery at the periphery of the lesions (as shown in Tables 7 and 8 and
(87) TABLE-US-00007 TABLE 7 Pathological evaluation criteria of damage and inflammatory infiltration of the terminal bronchioles Inflammatory cell infiltration Damage to the terminal of the terminal Score bronchiole wall bronchioles 0 The tissue structure The tissue structure is normal. is normal and no inflammatory cell infiltration is observed. 1 The tissue structure is Scattered inflammatory normal, accompanied by cell infiltration can be wall damage within observed in the tunica of the area, manifested externa of wall, which is as damage, regeneration not focal, and the number of bronchial of the inflammatory epithelium, edema of cells is less than 10. wall, degeneration or regeneration of tunica media muscular layer. 2 The tissue structure is Numerous scattered normal, accompanied by inflammatory cell wall damage in more infiltrations can be observed than of the area, in the tunica externa manifested as damage, of wall, which are focal, regeneration of single or multiple, bronchial epithelium, accumulating in less edema of wall, than of the area of the degeneration or wall. regeneration of the tunica media muscular layer. 3 The tissue structure is Diffuse inflammatory normal, accompanied by cell infiltration can be wall damage in more observed in tunica externa than of the area, of wall, accumulating manifested as damage, in more than of regeneration of the area of the wall, or bronchial epithelium, inflammatory cell infiltration edema of wall, can be observed in degeneration or tunica intima, tunica media. regeneration of the tunica media muscular layer, formation of tunica externa granuloma or fibrosis.
(88) TABLE-US-00008 TABLE 8 Pathological evaluation criteria of damage and inflammatory infiltration of the small pulmonary arterioles Inflammatory cell Damage to the small infiltration of the small Score pulmonary arterioles pulmonary arterioles 0 Normal small pulmonary Normal small pulmonary arterioles structure. arterioles structure. 1 Exfoliation of the Scattered inflammatory some endothelial cells. cell infiltration can be observed in the tunica externa of wall, which is not focal, and the number of the inflammatory cells is less than 10. 2 Exfoliation of the Numerous scattered endothelial cells, inflammatory cell degeneration, hyperplasia infiltrations can be observed or small focal in the tunica externa necrosis of the tunica of the wall, which are focal, media smooth single or multiple, muscle. accumulating in less than of the tunica externa of the wall. 3 Exfoliation of endothelial Diffuse inflammatory cells, degeneration, cell infiltration can be hyperplasia or small focal observed in the tunica necrosis of tunica externa of the wall, media smooth muscle, accumulating in more formation of tunica than of the area of the externa granuloma or fibrosis. wall, or inflammatory cell infiltration can be observed in the tunica media.
(89) TABLE-US-00009 TABLE 9 Pathological evaluation criteria of pulmonary fibrosis Fibrosis grading Ashcroft scoring criteria 0 Alveolar septum: No fibrotic lesions; Lung structure: Normal. 1 Alveolar septum: Solitary simple fibrotic change (The thickness of the alveolar septum increases, but is less than three times that of a normal lung); Lung structure: Partial enlargement of the alveolar space, small amount of exudate, and no fibrotic material. 2 Alveolar septum: Clear fibrotic change (The thickness of alveolar septum increases, but is more than three times that of a normal lung) forms into a small nodule, but is not contiguous; Lung structure: Partial enlargement of the alveolar space, small amount of exudate, and no fibrotic material. 3 Alveolar septum: Uninterrupted fibrosis can be observed in almost all alveolar walls in each high power field (The thickness of alveolar septum increases, but is more than three times that of a normal lung); Lung structure: Partial enlargement of the alveolar space, small amount of exudate, and no fibrotic material. 4 Alveolar septum: The alveolar septum is still visible; Lung structure: Solitary fibrotic nodules appear in the alveolar space (10% of high power field). 5 Alveolar septum: The alveolar septum is still visible; Lung structure: Fused fibrotic nodules appear in alveolar space (>10% and 50% of high power field), and lung tissue structure is severely damaged, but the structure still remains. 6 Alveolar septum: Visible, but almost nonexistent; Lung structure: Large uninterrupted fibrotic nodules (>50% of high power field), and framework of lung tissue is almost nonexistent. 7 Alveolar septum: No longer exists; Lung structure: The alveolar space is almost filled with fibrotic material, but there are still less than 5 vacuole-like structures. 8 Alveolar septum: No longer exists; Lung structure: Under high magnification, alveolar space is filled with the fibrotic tissue.
2.8 Data Analysis
(90) The meansd or meansem was calculated using the graphpad prism software, and the significant difference test was performed using t-test, one-way ANOVA and two-way ANOVA test. A significant difference between the two groups was considered at p<0.05.
3. Experimental Results
3.1 Basic Physiological Observation of the Animals During Administration
(91) All experimental animals showed no obvious physiological and behavioral abnormal changes during the administration.
3.2 Changes in Body Weight of all the Animals During the Experiment
(92) During the experiment, the animals in each group had a slight decrease in body weight in a short period of time (5-7 days). The body weight of all the animals gradually increased with the progress of the experiment (the results are shown in Table 10 and
(93) TABLE-US-00010 TABLE 10 Changes in body weight of the animals (Mean SEM) Day Group 1 3 5 7 9 11 13 Model (QD) 271.0 3.7 251.7 4.6 260.3 7.2 271.0 8.6 284.3 10.6 291.7 13.0 300.0 15.5 PFD-50mpk (BID) 279.4 2.3 261.8 3.9 265.0 6.4 282.9 7.4 298.4 8.0 312.6 8.8 321.7 8.3 Slit2D2(C386S)-HSA-1mpk 273.8 3.1 255.2 3.2 262.8 4.7 276.7 5.4 290.7 6.3 302.7 6.9 310.3 7.8 (Q2D) Slit2D2(C386S)-HSA-5mpk 272.7 1.8 251.8 2.3 245.0 5.0 258.3 4.4 272.5 5.0 284.3 5.9 292.2 5.6 (Q2D)
3.3 Results of the General Examination of Lung
(94) The changes in volume and wet weight of the left lung of the animals after perfusion with an equal amount of fixative are shown in Table 11. Compared with the model group, the left lung of the animals in each administration group was reduced in both volume and wet weight, but there was no significant difference. There was no significant difference in the volume and wet weight of the diseased lung tissue in each group. After two weeks of pulmonary fibrosis in the left lung, in each experimental group, the lung volume was reduced and the weight was correspondingly reduced, and there was no significant difference compared with the model group (results are shown in
(95) TABLE-US-00011 TABLE 11 Weight and volume of left lung (Mean SEM) Weight of the Volume of the Group left lung (mg) left lung (mm.sup.3) Model (QD) 1.27 0.20 1.50 0.17 PFD-50mpk (BID) 1.10 0.12 1.14 0.11 Slit2D2(C386S)-HSA-1mpk (BID) 1.20 0.03 1.27 0.02 Slit2D2(C386S)-HSA-5mpk (BID) 1.31 0.07 1.32 0.03
3.4 Pathological Evaluation of Injury of the Left Lung
(96) Histological observation of the diseased lung tissue showed significant lung injury with clear lung tissue boundaries (as shown in
3.5 Pathological Evaluation of the Pulmonary Fibrosis
(97) Pulmonary histological masson staining clearly showed the uniform fibrotic lesions and the distribution range of the lesions in the left lung (as shown in
(98) The pathological changes and degree of the pulmonary fibrosis in left lung were scored using Masson Trichrome staining Histological lesions include alveolar wall structural damage, thickening, inflammatory cell infiltration, collagen fiber deposition; alveolar cavity filled with heterogeneous inflammatory exudate, and fibrotic mass in some alveolar spaces. The normal structure of lung tissue in the severely damaged area completely disappeared and was replaced by fibrosis and inflammatory granuloma tissue. Each test compound group showed different effects in inhibiting fibrosis. Ashcroft scoring results indicated the inhibitory and remission effects of different test compounds on pulmonary fibrosis (as shown in
4. Conclusion
(99) Direct injection of BLM trachea successfully induced left unilateral pulmonary fibrosis. The area of injury in the left lung of all the animals caused by BLM was uniform, accounting for about 80% of the section of left lung. There were no significant differences between the experimental groups, which suggested that the BLM-induced left lung pulmonary fibrosis model was stable.
(100) Continuous administration of positive control drug (PFD) for 14 days showed significant inhibition on the progression of pulmonary fibrosis.
(101) Intravenous administration of Slit2D2(C386S)-HSA every other day for 2 weeks showed significant inhibition on the progression of pulmonary fibrosis, with a dose-dependent trend. The Slit2D2 (C386S)-HSA 5 mg/kg dose group was superior to the positive compound (PFD) in inhibiting pulmonary fibrosis.
(102) The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and scope of the present invention, are intended to be included within the scope of the present invention.