COMPOSITION OF DRUG TARGETS AND METHOD OF USING THEREOF

Abstract

A composition of drug targets and the use the method of using thereof. The composition comprises a vector and a drug using FKBP10 and PCOLCE genes and/or the encoded proteins thereof as drug targets.

Claims

1. A composition of drug targets, comprising: a vector, and a drug using FKBP10 and PCOLCE genes and/or the encoded proteins thereof as drug targets.

2. The composition of drug targets according to claim 1, wherein the drug using FKBP10 and PCOLCE genes and/or the encoded proteins thereof as drug targets is an inhibitor for FKBP10 and PCOLCE genes and/or the encoded proteins thereof.

3. The composition of drug targets according to claim 1, wherein the drug using FKBP10 and PCOLCE genes and/or the encoded proteins thereof as drug targets is a neutralizing antibody, a small molecule drug, a small nucleotide, a microRNA, an antisense molecule or a polypeptide for interfering with the expression and function of FKBP10 and PCOLCE genes and/or the encoded proteins thereof.

4. The composition of drug targets according to claim 3, wherein the interfering mode comprises simultaneously interfering with KBP10 and PCOLCE genes and/or the encoded proteins thereof, or first interfering with KBP10 gene and/or the encoded protein thereof and then interfering with PCOLCE gene and/or the encoded protein thereof.

5. The composition of drug targets according to claim 1, wherein the drug using FKBP10 and PCOLCE genes and/or the encoded proteins thereof as drug targets is administered by local injection or smearing.

6. The composition of drug targets according to claim 1, wherein the vector is a histidine polypeptide, a lysine polypeptide, a branched histidine-lysine copolymer polypeptide, a cationic polymer, silicon nanoparticles, a lipid vector, or a viral vector.

7. A method of using the composition of drug targets according to claim 1, wherein the composition is used for preparing a medicament for treating fibrosis-related diseases.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows a comparison of differences between FKBP10 and PCOLCE expression in human hypertrophic scar and normal skin;

[0025] FIG. 2 shows knock downing FKBP10 and/or PCOLCE expression can inhibit the expression of fibrosis markers in human hypertrophic scar fibroblasts;

[0026] FIG. 3 shows interfering with human FKBP10 and/or PCOLCE using a small nucleotide can inhibit the activity of human hypertrophic scar fibroblasts;

[0027] FIG. 4 shows interfering with mouse FKBP10 and/or PCOLCE using a small nucleotide can inhibit the formation of mouse hypertrophic scars; wherein, A: HE staining of human hypertrophic scar and normal skin; B: HE staining of mouse models in various interfering modes; C: Masson staining of mouse models in various interfering modes; D: a statistic diagram of collagen content in Masson stained mouse models;

[0028] FIG. 5 shows interfering with mouse FKBP10 and/or PCOLCE using a small nucleotide can inhibit the expression of fibrosis markers in mice's skin.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is further described below in conjunction with specific Examples. It should be understood that these Examples are only used to illustrate the present invention rather than to limit the scope of the present invention. In addition, it should be understood that, after reading the content taught by the present invention, persons skilled in the art can make various changes or modifications to the present invention, and these equivalents also fall within the scope defined by the appended claims of the present application.

[0030] There are many ways to interfere with the expression and function of FKBP10 and PCOLCE genes and/or the encoded proteins thereof, for example, using a neutralizing antibody, a small molecule drug, a small nucleotide, a microRNA, or a polypeptide. Taking a small nucleotide drug as an example and human hypertrophic scar fibroblasts (HHSF) as target cells, the Examples demonstrate that interfering with FKBP10 and PCOLCE expression can inhibit the activity of HHSF, and decrease the expression of fibrosis-related markers. In addition, by establishing mouse hypertrophic scar models and injecting a small nucleotide into local skin, it has been verified that interfering with FKBP10 and PCOLCE can inhibit the formation of mouse hypertrophic scars.

Example 1

[0031] 1. Experimental Materials

[0032] 1.1 Small Nucleotide Sequence

[0033] Small nucleotide (Small interfering RNA, siRNA): It is a small RNA molecule (˜21-25 nucleotides) processed by Dicer (an enzyme specific for double-stranded RNA in the RNAase III family). SiRNA is a main member of siRISC, which stimulates silencing of the complementary target mRNA. RNA interference (RNAinterference, RNAi) refers to specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsRNA), which leads to silencing of the expression of the target gene and loss of corresponding functional phenotype.

[0034] Its sequence for human FKBP10mRNA:

TABLE-US-00001 Sense strand: 5′-GAAGGAAGAUUAUCAUCCCUCCAUU-3′; Antisense strand: 5′-AAUGGAGGGAUGAUAAUCUUCCUUC-3′;

[0035] Its sequence for mouse FKBP10 mRNA:

TABLE-US-00002 Sense strand: 5′-CCACACCUACAAUACCUAUTT-3′; Antisense strand: 5′-AUAGGUAUUGUAGGUGUGGTT-3′;

[0036] Its sequence for human PCOLCE mRNA:

TABLE-US-00003 Sense strand: 5′-CCUUCCUCCAGAGAGCUUU-3′; Antisense strand: 5′-GGAAGGAGGUCUCUCGAAA-3′;

[0037] Its sequence for mouse PCOLCE mRNA:

TABLE-US-00004 Sense strand: 5′-CCUGGCAACCAAGUGACUU-3′; Antisense strand: 5′-GGACCGUUGGUUCACUGAA-3′

[0038] 1.2 Method for Formulating a Solution of Small Nucleotide/Histidine-Lysine Polymer (HKP) Nanoparticles/Methyl Cellulose

[0039] The histidine-lysine polymer (HKP), which is used as a vector for siRNA transfection, has a lysine skeleton, including a branched chain containing multiple histidine, lysine or aspartic acid. HKP was dissolved in DEPC-treated water to form a DEPC aqueous solution. Then the DEPC aqueous solution was mixed with a siRNA (purchased from Shanghai Jima Pharmaceutical

[0040] Technology Co., Ltd.) aqueous solution at a mass ratio of 4:1 and a volume ratio of 1:1 to form nanoparticles having an average diameter of 150-200 nm. The resulting HKP-siRNA aqueous solution was translucent without any obvious sediment aggregation, and could be stored at 4° C. for at least three months.

[0041] 1.3 Method for Formulating Small Nucleotide Injections

[0042] Formulation of small nucleotide injections for in vivo experiments: Specially modified siRNA (purchased from Ruibo Biotechnology Co., Ltd.) and HKP were dissolved with a DEPC-5% glucose solution to prepare siRNA and HKP aqueous solutions, respectively. Then, the HKP aqueous solution was mixed with the siRNA aqueous solution at a mass ratio of 4:1 and a volume ratio of 1:1 to form nanoparticles having an average diameter of 150-200 nm. The resulting HKP-siRNA aqueous solution was translucent without any obvious sediment aggregation, and could be stored at 4° C. for at least three months.

[0043] 2. Experimental Methods

[0044] 2.1 Extraction and Cultivation of Human Fibroblasts

[0045] Clinical specimens (human hypertrophic scars, keloids or normal skin) were soaked with Dispase II (2 mg/ml, Life Technologies, ThermoFisher) overnight at 4° C., before they were peeled and removed of the epidermis. The tissues were minced in a sterile environment, soaked with 4 mg/ml collagenase, and digested with a shaker at 37° C. for 2-4 hours. The cell suspension was filtered with a filter screen, and centrifuged at 1500 rpm for 5 min to remove the supernatant. The cell sediments were re-suspended in a culture medium, and then inoculated to a DMEM medium. This medium was changed every 2 days for routine cultivation.

[0046] 2.2 Establishment of Mouse Hypertrophic Scar Models

[0047] 12-week-old C57/BL6 mice were anesthetized to perform a longitudinal incision of 1 cm on their back skin, under which micro-permeable capsules (Alzet Model 1004; Durect Corp., Cupertino, Calif., USA) filled with a Bleomycin solution (2.8 mg/ml) were embedded, and the incision was sutured. After 56 days, the mice were euthanized and sampled.

[0048] 2.3 Small Nucleotide Injections

[0049] On the first day when scars were formed on the mice, small nucleotide injections through intradermal injection was started. The mice in the control group, FKBP10 siRNA alone administration group, and PCOLCE siRNA alone administration group were injected once every 3 days until sampling on the 56th day of model establishment. As for the FKBP10 siRNA+PCOLCE siRNA simultaneous administration group, the formulated injections have a final FKBP10 siRNA concentration of 200 μg/ml, and a final PCOLCE siRNA concentration of 50 μg/ml, and each of these two drugs was combined with HKP to prepare HKP-siRNA solutions for mixed injection once every 3 days until sampling on the 56th day of model establishment. As for the FKBP10 siRNA+(24 h) PCOLCE siRNA administration group, FKBP10 siRNA (200 μg/ml) was injected on the first day, PCOLCE siRNA (50 μg/ml) was injected 24 hours later, and the two drugs were alternately injected every 3 days until sampling on the 56th day of model establishment.

[0050] 2.4 siRNA Transfection of In Vitro Cultured Cells

[0051] When the cell density reached about 70%, the medium in which the cells were cultivated was changed, and added with the HKP-siRNA aqueous solution (refer to Section 1.2 for the method). For the cells in the control group, FKBP10 siRNA alone administration group, and PCOLCE siRNA alone administration group, the final siRNA concentration in the culture medium is 50 nM, and the cells were tested after 24 hours of cultivation. For the cells in the FKBP10 siRNA+PCOLCE siRNA simultaneous interfering group, the final FKBP10 siRNA concentration in the culture medium is 50 nM and the final PCOLCE siRNA concentration in the culture medium is 25 nM. Each of FKBP10 siRNA and PCOLCE siRNA was combined with HKP to prepare HKP-siRNA solutions before they were mixed in the culture medium. The cells were tested after 24 hours of cultivation. For the cells in the FKBP10 siRNA+(24 h) PCOLCE siRNA interfering group, the cells were first cultivated in a culture medium with a final FKBP10siRNA concentration of 50 nM, then 24 hours later changed to be cultivated in a culture medium with a final PCOLCE siRNA concentration of 50 nM, and later tested after 24 hours of cultivation.

[0052] 2.5 Real-Time Quantitative PCR

[0053] Total RNA of tissues or cells was extracted by Trizol (Invitrogen, Grand Island, N.Y., USA), and RNA concentration and purity were measured by ultraviolet spectrophotometry (ND-1000, Thermo, Rockford, Ill., USA). The extracted total RNA was subjected to reverse transcription reaction by means of a RT-PCR kit (TaKaRa, Shiga, Japan) and ABI HT7900 PCR instrument (Applied Biosystems, Foster City, Calif., USA) to synthesize cDNA. The above cDNA served as a template for real-time quantitative PCR.

[0054] In a 200 reaction system:

[0055] MIX (SYBR Premix Ex Taq): 10 μl;

[0056] Upstream primer: 0.4 μl;

[0057] Downstream primer: 0.4 μl;

[0058] ROX II: 0.4 μl;

[0059] cDNA: 20;

[0060] Deionized water: 6.8 μl.

[0061] Each reaction tube was prepared, briefly centrifuged to mix evenly, and then placed in a PCR reactor (Applied Biosystems). Reaction conditions: after hot start at 95° C. for 10 seconds, running the process for 40 cycles as 95° C. for 15 seconds, 60° C. for 30 minutes, 72° C. for 3 minutes. When the reaction was completed, the results were analyzed with an analysis software using ΔΔCT Relative Quantitation method. Each sample was configured with three parallel wells, and the GADPH primer served as an internal control.

[0062] 2.6 Immunohistochemistry/Fluorescence Staining

[0063] Clinical specimens and animal tissues were sampled, routinely fixed, dehydrated, and embedded in paraffin, and prepared into 6 μm sections. After dewaxing, these sections were blocked with 1% sheep serum, and incubated with primary antibodies at 4° C. overnight. Then the sections were washed with BSA, and incubated with secondary antibodies comprising horseradish peroxidase or labeled with fluorescence at 37° C. for 2 hours. Colors were developed and pictures were taken.

[0064] 2.7 Detection of CCK-8 Cell Activity

[0065] The proliferation activity of cells cultivated in vitro was tested by means of the CCK-8 method. The CCK-8 kit was purchased from Dojindo (Tokyo, Japan), and the experimental procedures were carried out in strict accordance with the kit instructions.

[0066] 3. Experimental Results

[0067] 3.1 Comparison of Differences Between FKBP10 and PCOLCE Expression in Human Hypertrophic Scar and Normal Skin

[0068] Clinical samples (human hypertrophic scar and normal skin) were subjected to immunohistochemical detection. The immunohistochemical results showed that the expressions of FKBP10 and PCOLCE in human hypertrophic scars were significantly higher than those in normal skin (p<0.001) (FIG. 1).

[0069] 3.2 Knocking Down FKBP10 and/or PCOLCE Expression can Inhibit the Expression of Fibrosis Markers in Human Hypertrophic Scar Fibroblasts

[0070] Human hypertrophic scar fibroblasts were isolated and cultivated, in which FKBP10 and/or PCOLCE were knocked down by means of RNAi technology, and the mRNA levels of fibrosis markers COL1A, COL3A, FN, α-SMA and TGF-β1 MRNA were detected by real-time quantitative PCR (q-PCR). The q-PCR results showed that knocking down FKBP10 and/or PCOLCE could significantly reduce the expression of fibrosis markers COL1A, COL3A, FN, α-SMA and TGF-β1 (p<0.001). Among them, the cells treated by two methods, namely, simultaneously interfering with two targets FKBP10 and PCOLCE, and first interfering with FKBP10 target and 24 hours later interfering with PCOLCE target, had lower expression of fibrosis markers than the cells in the group where FKBP10 or PCOLCE was interfered with alone. In other words, interfering with dual targets of FKBP10 and PCOLCE achieved better effect than interfering with one of the two targets alone (FIG. 2).

[0071] 3.3 Interfering with Human FKBP10 and/or PCOLCE Using a Small Nucleotide can Inhibit the Activity of Human Hypertrophic Scar Fibroblasts

[0072] Human hypertrophic scar fibroblasts were isolated and cultivated, in which the expression of FKBP10 and/or PCOLCE was interfered with using a small nucleotide. The proliferation activity of human hypertrophic scar fibroblasts was detected by means of the CCK-8 method. The results showed that after interfering for 5d and 7d, the proliferation activity of the cells with FKBP10 and/or PCOLCE knocked down was significantly lower than that in the control group. Meanwhile, the cells treated by two methods, namely, simultaneously interfering with two targets FKBP10 and PCOLCE, and first interfering with FKBP10 target and 24 hours later interfering with PCOLCE target, had lower proliferation activity than the cells in the group where FKBP10 or PCOLCE was interfered with alone. In other words, interfering with dual targets of FKBP10 and PCOLCE achieved better effect than interfering with one of the two targets alone (FIG. 3).

[0073] 3.4 Interfering with Mouse FKBP10 and/or PCOLCE Using a Small Nucleotide can Inhibit the Formation of Mouse Hypertrophic Scars

[0074] Classic mouse hyperplastic scar models were established to simulate pathological changes of human hypertrophic scar (FIGS. 4A-C). HE and Masson staining were performed on the sampled mouse scars. The results showed that after interfering with FKBP10 and/or PCOLCE, the characteristics of skin hypertrophic scars (these characteristics include: thickening of epidermis and dermis, reduction of skin accessory organs, increased collagen and disordered arrangement, etc.) were significantly suppressed (p<0.001). Among them, the two methods, namely, simultaneously interfering with two targets FKBP10 and PCOLCE, and first interfering with FKBP10 target and 24 hours later interfering with PCOLCE target, achieved significantly better effect than interfering with FKBP10 or PCOLCE alone (p<0.05).

[0075] 3.5 Interfering with Mouse FKBP10 and/or PCOLCE Using a Small Nucleotide can Inhibit the Expression of Fibrosis Markers in Mice's Skin

[0076] A small nucleotide was used to interfere with the expression of FKBP10 and/or PCOLCE in the mouse model, and the skin samples were subjected to q-PCR detection. The results showed that interfering with FKBP10 and/or PCOLCE could significantly reduce the expression of collagen I, collagen III, fibronectin, α-SMA and TGF-β1 (p<0.001). Among them, the skin treated by the two methods, namely, simultaneously interfering with two targets FKBP10 and PCOLCE, and first interfering with FKBP10 target and 24 hours later interfering with PCOLCE target, had lower expression of fibrosis markers than the skin in the group where FKBP10 or PCOLCE was interfered with alone. In other words, interfering with dual targets of FKBP10 and PCOLCE achieved better effect than interfering with one of the two targets alone (FIG. 5).