Pharmaceutical application of PMEPA1-targeted ginkgo tRFs in asthma treatment

Abstract

The invention relates to a pharmaceutical application of ginkgo tRFs targeting PMEPA1 in treatment of asthma. The PMEPA1 is an important target which is screened out through information analysis and experimental verification and participates in asthma attack, is highly expressed in asthma and is in positive correlation with airway remodeling and airway inflammation. The tRFs adopting the gingko traditional Chinese medicine can significantly inhibit PMEPA1 and relieve collagen deposition, glycogen accumulation and airway inflammation of asthma mice. The invention prompts that PMEPA1 can become a new target for asthma pharmacy, and tRFs and pharmaceutical compositions derived from ginkgo traditional Chinese medicines can become brand new medicines for treating asthma. The medicine comprises the RNA molecule and a pharmaceutically acceptable carrier, a diluent and / or an excipient. The invention is helpful to research and development of asthma treatment medicines, and provides reference for research and development of other respiratory system disease treatment medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of asthma treatment, and particularly relates to the pharmaceutical use of ginkgo tRFs targeting PMEPA1 in asthma treatment. BACKGROUND

[0002] Bronchial asthma (asthma for short) is a chronic respiratory disease that seriously affects human health at all ages. According to the global burden of disease results in 2019: there are more than 300 million people with asthma in the world, and the incidence rate is showing a rising trend year by year. The asthma prevalence rate of people over 20 years old in China is 4.2%, and the total number of people over 20 years old with asthma is about 45.79 million. The results of epidemiological survey of risk factors for asthma prevalence and incidence in China show that the asthma prevalence rate of adolescents and adults aged 14 and above in China is 1.24%, of which severe asthma accounts for 7.1%, and severe refractory asthma accounts for 5.99%. The disease burden brought by asthma is one of the main sources of medical burden of chronic airway diseases.

[0003] Asthma is mainly manifested as airway inflammation, airway remodeling and airway hyperresponsiveness. The drugs currently used in clinical treatment of asthma include hormones, β-receptor agonists, targeted preparations, etc., but not all asthma patients can benefit from them. Airway remodeling is one of the difficulties that need to be overcome in asthma. The pathological manifestations of airway remodeling are airway pericollagen deposition, fibrosis aggravation, abnormal thickening of smooth muscle layer, etc. Airway remodeling not only occurs in the early stage of asthma, but also is one of the main reasons for lung function deterioration, and early intervention is beneficial to the long-term quality of life and prognosis of asthma patients. There is currently a lack of drug treatment programs for airway remodeling in clinical practice. Therefore, the design and synthesis of drugs targeting airway remodeling in asthma have important research value and clinical application prospect.

[0004] Transfer RNA (tRNA) is a key element of the protein synthesis machinery in cells. Recent studies have shown that tRNA in cells can also be specifically cleaved into 18-35mer fragments to exert a wide range of gene expression regulation functions, thereby regulating the physiological and pathological states of the body (such as cancer cell proliferation and invasion, Cell, 2015, 161, 790). Small nucleic acid drugs are one of the most concerned frontiers in the field of biological medicine today, and their main mechanism is to inhibit the expression of target proteins through RNA interference (RNAi) to achieve the purpose of treating diseases. Studies have shown that exogenous plant-derived nucleic acid fragments can also target RNA transcription or post-transcriptional processes to regulate biological processes. Ginkgo is a lung meridian, has the effects of astringing lung qi and relieving asthma and cough, and has certain effects on lung diseases such as lung deficiency cough and asthma. Therefore, it is feasible to use active fragments in ginkgo for the treatment of diseases. SUMMARY

[0005] Technical problems solved: The present application provides a pharmaceutical use of targeting PMEPA1 in asthma treatment drugs. The applicant previously screened PMEPA1 as an important target involved in the pathogenesis of asthma. Further determination of tRFs fragments in Ginkgo biloba can reduce the pathological phenotype of airway structural cells in asthma by inhibiting PMEPA1, thereby improving airway inflammation, high mucus secretion and airway remodeling. This can provide a theoretical basis for its application in the treatment of asthma.

[0006] Technical solutions: PMEPA1 as a target for the preparation or screening of asthma treatment drugs.

[0007] The asthma includes asthma of different age groups, severity and different inflammatory and immune phenotypes.

[0008] The application of Ginkgo biloba RNA molecules regulating PMEPA1 protein expression in the preparation of asthma treatment drugs.

[0009] A drug for treating asthma, the effective component contains a transfer RNA molecule fragment of Ginkgo biloba traditional Chinese medicine.

[0010] The sequence of the above-mentioned transfer RNA molecule fragment of Ginkgo biloba traditional Chinese medicine is a functional variant or a homolog thereof containing SEQ ID NO. 1.

[0011] The above-mentioned RNA molecule is a double-stranded RNA molecule, which is selected from any antisense sequence or a functional variant or a homolog thereof containing SEQ ID NO. 1, and any complementary sense sequence of SEQ ID NO. 2.

[0012] The above-mentioned transfer RNA molecule fragment of Ginkgo biloba traditional Chinese medicine or a functional variant or a homolog thereof, wherein the nucleotide sequence of the antisense strand of the double-stranded RNA molecule or a functional variant or a homolog thereof is as shown in SEQ ID NO. 1, and the nucleotide sequence of the sense strand of the double-stranded RNA molecule or a functional variant or a homolog thereof is as shown in SEQ ID NO. 2.

[0013] Preferably, the above-mentioned double-stranded RNA molecule or a functional variant or a homolog thereof contains a 3' overhang.

[0014] The nucleotide sequence of the antisense strand and / or the sense strand of the double-stranded RNA molecule or the functional variant or homolog thereof comprises at least one chemically modified nucleotide; wherein the chemical modification is at least one of 1-methyl, 2-methyl, 5-methyl, 7-methyl, N2-methyl, N6-methyl, N2,N2-dimethyl, 2'-O-methyl, N6-isopentenyl, 2-methylthio-N6-isopentenyl, N6-threonylcarbamoyl, N6-methyl-N6-threonylcarbamoyl, 2-thio, 4-thio, N4-acetyl, 5-formyl, 3-(3-amino-3-carboxypropyl), 5-methoxy, 5-oxoacetic acid, 5-oxoacetic acid methyl ester, 5-methoxycarbonylmethyl, 5,2'-O-dimethyl, 5-methoxycarbonylmethyl-2'-O-methyl, 5-methoxycarbonylmethyl-2-thio, 5-aminomethyl-2-thio, 5-methylaminomethyl, 5-methylaminomethyl-2-thio, 5-carbamoylmethyl, 5-carbamoylmethyl-2'-O-methyl, 5-carboxymethylaminomethyl, 5-carboxymethylaminomethyl-2'-O-methyl, 5-carboxymethylaminomethyl-2-methyl, 5-taurinomethyl, and 5-taurinomethyl-2-thio.

[0015] Preferably, the above-mentioned drug further comprises a nucleic acid stabilizer.

[0016] Specifically: (1) Bioinformatics screening found that PMEPA1 is a target involved in asthma airway remodeling. (2) An in vitro TGF-β1-induced airway epithelial cell model was established to verify the expression of PMEPA1 and its relationship with airway remodeling; (3) A house dust mite-induced asthma mouse model was established to determine the therapeutic effect of targeting PMEPA1 on asthma.

[0017] The experimental methods of the study mainly include the following parts:

[0018] 1. Screening of asthma targets

[0019] Through the existing GEO data set, the screening of asthma airway remodeling targets was first performed. The expression of PMEPA1 was verified at the cellular and animal levels.

[0020] 2. Cell function verification of target

[0021] Primary human airway smooth muscle cells (ASMC) were cultured in vitro, and CCK8 method was used to detect the effect of PMEPA1 inhibition on cell proliferation. Human bronchial epithelial cell line (BEC) was cultured in vitro, and scratch test was used to verify the effect of PMEPA1 on airway epithelial cell migration in airway remodeling, and WB was used to verify the effect of PMEPA1 on epithelial-mesenchymal transition (EMT) related indicators in airway remodeling.

[0022] 3. Animal level verification of target

[0023] Control group, asthma group and ginkgo treatment group mouse models were constructed respectively. Mouse lung tissues were collected for subsequent hematoxylin-eosin (HE), periodic acid-schiff (PAS), masson staining and immunohistochemistry, and the levels of airway inflammation, glycogen accumulation, collagen deposition and PMEPA1 protein expression in mouse lung tissues were evaluated respectively.

[0024] 4. Statistical analysis method

[0025] The animal experiment data was expressed as mean ± standard error (±SEM), and the data of in vitro experiment was expressed as mean ± standard deviation (±SD), and statistical analysis was performed using Graphpad Prism 9.0. The comparison between two groups used t test. The comparison among multiple groups used one-way analysis of variance. P<0.05 represented that the difference had statistical significance.

[0026] We first screened and confirmed that PMEPA1 was highly expressed in asthma and was related to airway remodeling. Through the matching of the traditional Chinese medicine tRNA database we screened, we found that tRF_11 from ginkgo could be highly complementary to PMEPA1. In vitro, tRF_11 from ginkgo could significantly inhibit the expression of PMEPA1, inhibit the proliferation of ASMC, BEC migration and EMT. In vivo, the airway inflammation, airway remodeling and epithelial glycogen accumulation in the ginkgo tRF_11 treatment group were significantly reduced. The above results mean that PMEPA1 may become a new target for designing asthma drugs, and ginkgo tRFs can become a new type of drug for treating asthma. Specifically, ginkgo tRFs can be synthesized, appropriately modified, and wrapped with appropriate carriers such as nanoparticles, liposomes, etc. to form drugs, and asthma can be treated by nasal drops, oral administration, etc.

[0027] Beneficial effects: The application proves that PMEAP1 is related to airway remodeling as a new target in asthma at in vivo and in vitro levels, and proves the influence of tRFs fragments from ginkgo on airway remodeling and airway inflammation by targeting PMEPA1, and reveals the potential of targeting PMEPA1 and small nucleic acid fragments from traditional Chinese medicine in treating asthma. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Screening and verification of asthma airway remodeling related targets. Wherein A is the screening result of GEO database target; B is the expression of PMEPA1 in human airway smooth muscle cells and airway epithelial cells after TGF-β1 induction; C is the expression of PMEPA1 and TGF-β1 in HDM induced asthma mouse lung tissue.

[0029] Figure 2 Relationship between PMEPA1 and airway remodeling. Wherein A is the influence of targeting PMEPA1 on airway smooth muscle proliferation; B is the influence of targeting PMEPA1 on human bronchial epithelial cell migration and EMT. Wherein si-YX is ginkgo tRF_11, and P3 is the siRNA of PMEPA1.

[0030] Figure 3 Influence of targeting PMEPA1 on airway remodeling, airway inflammation and mucus hypersecretion in asthma mice. A is the schematic diagram of asthma mouse modeling; B is the HE staining of lung tissue of different groups of mice; C is the PAS staining of mouse lung tissue; D is the Masson staining of lung tissue; E is the immunohistochemistry of lung tissue PMEPA1

[0031] In all column charts of statistical analysis, *, **, ***, **** represent P<0.05, P<0.01, P<0.001, P<0.0001, respectively. DETAILED DESCRIPTION

[0032] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, "comprising" means including, but not limited to, the recited elements. "Consisting of" means formed from the recited elements.

[0033] The application discloses that PMEPA1 is one of the new treatment targets of asthma, PMEPA1 is screened out through comprehensive bioinformatics analysis results, and through further in vivo and in vitro verification, it is found that PMEPA1 is highly expressed in asthma, and is positively correlated with asthma airway remodeling.

[0034] It is further disclosed a tRF fragment of Ginkgo biloba origin that can be applied in the treatment of asthma by targeting PMEPA1, the RNA molecule to be administered according to the present application can be naturally occurring, modified or artificially synthesized according to the sequences disclosed in the present application. The sequence of the RNA molecule of the present application is of about 10 to 200 nucleotides in length, which can be considered as a small RNA molecule. Preferably, the sequence of the RNA molecule is of about 10 to about 50 nucleotides in length.

[0035] The RNA molecule of the present application or a functional variant thereof or a homologue thereof is selected from the sequence of SEQ ID NO. 1. The term "functional variant" of the RNA molecule refers to a molecule substantially similar to the RNA molecule having one or more sequence alterations that do not affect the biological activity or function of the RNA molecule. Sequence alterations that do not affect the functional properties of the resulting RNA molecule are well known in the art. For example, nucleotide changes that result in alterations in the -5'-end and -3'-end portions of the molecule are not expected to change the activity of the polynucleotide.

[0036] In particular, the functional variant of the RNA molecule has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity to the non-variant RNA molecule according to the present application.

[0037] The term "homologue" as used herein refers to a nucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the RNA molecule according to the present application. In one embodiment, the homologue of the RNA molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity to the RNA molecule.

[0038] The RNA molecule of the present application can be administered in the form of a pharmaceutical composition comprising the RNA molecule and at least one pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a colorant, a surfactant, a gene delivery vehicle and a preservative. The pharmaceutical composition can exist in a solid, semi-solid or liquid form, preferably in a liquid form. The pharmaceutical preparation can be a liposome lyophilized powder; a polypeptide nano-lyophilized powder; a spray; a tablet; etc. The pharmaceutical composition can comprise an additional pharmaceutically effective ingredient. The skilled person is able to select a suitable pharmaceutically acceptable excipient depending on the form of the pharmaceutical composition and knows the methods of preparing a pharmaceutical composition and is able to select a suitable method of preparing a pharmaceutical composition depending on the kind of the pharmaceutically acceptable excipient and the form of the pharmaceutical composition.

[0039] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and processes. However, the scope of the present invention is not limited to the following embodiments. Conditions and methods not specified in the following embodiments are performed according to conventional methods.

[0040] All reagents used in the following examples are analytical grade and can be purchased from legitimate sources.

[0041] Table 1. Ginkgo tRF_11 that can target PMEPA1 by artificial synthesis according to the present invention

[0042]

[0043] Table 2. List of abbreviations for modification symbols in tRNA sequences

[0044]

[0045]

[0046] The antisense and sense sequences of SEQ ID NO.1 shown in Table 1 can be artificially synthesized according to the present invention. The derived sequence fragments are divided into two categories: the first category is 5'-tRFs, which are fragments of 2-35 nucleotides in length formed by cleaving the 5' end of the mature tRNA sequence at the D loop, D loop arm, anticodon loop, or anticodon loop arm; the second category is 3'-tRFs, which are fragments of 2-35 nucleotides in length formed by cleaving the 3'-CCA end of the mature tRNA sequence at the T loop, T loop arm, anticodon loop, or anticodon loop arm. Each sense sequence, together with the corresponding antisense sequence, forms a double-stranded RNA molecule.

[0047] Furthermore, the RNA molecule of the present invention may contain a 3' overhang, preferably a 3' overhang containing 2 nucleotides. Providing a 3' overhang improves the stability of the RNA molecule.

[0048] Example 1: Screening and Validation of Targets in Asthma

[0049] First, the GEO database was used to screen for targets in asthma, and PMEPA1 was found to be highly expressed in asthma, such as... Figure 1 As shown in Figure A.

[0050] Constructing an in vitro airway remodeling-related cell structure model

[0051] Human BEC was cultured in 1640 complete medium with 10% serum concentration (45 mL basal 1640 medium + 5 mL serum + 500 μL double antibody). Human ASMC was cultured in SMCM medium with 2% serum concentration. After the cell density reached 80-90%, it was starved for 6-8 hours, and then intervened with TGF-β1 for 24 hours (TGF-β1 is a key factor involved in asthma airway remodeling).

[0052] Construction of asthma mouse model

[0053] Wild-type BALB / c female mice, 6-8 weeks old, weighing about 18-20 g. Asthma mice were sensitized by intranasal instillation of 25 μg HDM, 5 days a week. The control group of mice was given PBS throughout the process.

[0054] Verification of PMEPA1 expression in vivo and in vitro

[0055] The cell and mouse lung tissue proteins after modeling were extracted, and the PMEPA1 protein level was verified by Western Blot. The results are shown in Figure 1 : B and Figure 1 : C.

[0056] PMEPA1 is highly expressed in the lung tissue of asthma mice, ASMC and BEC after TGF-β1 intervention.

[0057] Example 2 Relationship between PMEPA1 and airway remodeling

[0058] CCK8 assesses the effect of PMEPA1 on ASMC proliferation

[0059] Logarithmic growth period human ASMC and BEC were taken and inoculated in 96-well plates for incubation, with 8000 cells per well, divided into four groups (control group (Control), TGF-β1, P3 (TGF-β1 + si-PMEPA1), si-YX (TGF-β1 + ginkgo tRF_11)). The P3 and si-YX groups were intervened with PMEPA1 siRNA fragment (si-PMEPA1) and ginkgo tRF_11 for 6-8 hours before TGF-β1 induction. After the cell intervention was completed, CCK8 solution prepared with blank medium at a ratio of 1:9 (10 μL CCK8 solution + 90 μL medium) was added to each well (note to prevent the generation of bubbles to avoid affecting the determination of OD value), and incubation was continued for about 1-2 hours. The absorbance value (OD value) was detected at 450 nm wavelength on the enzyme-labeled detector. Cell viability percentage (%) = (treatment group OD value-blank hole OD value) / (control group OD value-blank hole OD value) x 100%. As shown in Figure 2: A shows that, after the intervention of si-PMEPA1 and Ginkgo tRF_11, the over-proliferation of smooth muscle cells induced by TGF-β1 can be reduced.

[0060] Scratch test to evaluate the effect of PMEPA1 on BEC migration

[0061] As shown in Example (1) above, the cells were divided into 4 groups. After the cell confluence reached nearly 100%, the cells were first treated with siRNA fragments of PMEPA1 (si-PMEPA1) and Ginkgo tRF for 6-8 hours. Then the cells in the well plate were scratched, and after washing the cells with PBS, TGF-β1 was added for intervention. The cell migration at 0h, 8h and 24h was recorded under a microscope, respectively. The results are shown in Figure 2 : B shows that, after the intervention of si-PMEPA1 and Ginkgo tRF, the migration of BEC induced by TGF-β1 can be reduced.

[0062] WB to verify the effect of PMEPA1 on EMT

[0063] As shown in Example (1) above, the cells were divided into 4 groups. After the intervention, the cells were extracted, and the protein level of N-cadherin related to EMT was detected by Western Blot, and the results are shown in Figure 2 : C shows that, after the expression of PMEPA1 is inhibited, the EMT process can be reduced.

[0064] Example 3 Effect of targeting PMEPA1 on airway remodeling, airway inflammation and mucus hypersecretion in asthmatic mice

[0065] Construction of mouse model

[0066] The mice in the treatment group were pre-treated with Ginkgo tRF_11 one hour before HDM stimulation, and the mice in the asthma group and the control group were treated as before, and the results are shown in Figure 3 : A.

[0067] (2) Lung histopathology detection

[0068] The right lower lung tissue of the mouse was fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned and stained. HE staining was used to evaluate airway inflammation, and PAS staining was used to evaluate airway epithelial mucin secretion. Four images were randomly selected using a double-blind method for scoring. The HE staining score standard is: 0 points: normal; 1 point: few inflammatory cells; 2 points: inflammatory cell circle, thickness is 1 cell; 3 points: inflammatory cell circle 2-4 cells deep; 4 points: inflammatory cell circle > 4 cells deep. PAS staining score is based on the following criteria: 0 points: normal; 1 point: the proportion of stained cells is less than 25% of the entire epithelium; 2 points: 25-50%; 3 points: 0-75%; 4 points: > 75%. Masson staining was used to evaluate collagen deposition. The results are shown in Figure 3: B-D showed that the lung inflammation, mucus secretion and collagen deposition were significantly inhibited after the treatment of Ginkgo tRF_11. The expression and distribution of PMEPA1 protein in lung tissue were observed by immunohistochemistry. The results were shown in Figure 3 : E showed that the level of PMEPA1 was significantly reduced in the Ginkgo tRF_11 treatment group.

[0069] Conclusion: PMEPA1 is highly expressed in asthma and is associated with airway remodeling, while exogenous Ginkgo tRF_11 can reduce airway inflammation, mucus secretion and airway remodeling by inhibiting the expression of PMEPA1. Therefore, the PMEPA1 target has good application prospects for the development of asthma drugs.

Claims

1. Use of PMEPA1 as a target in the preparation or screening of drugs for treating asthma.

2. Use according to claim 1, characterized in that, The asthma includes asthma of different ages, severity, and different inflammatory and immune phenotypes.

3. Use of Ginkgo RNA molecules modulating the expression of PMEPA1 protein in the preparation of drugs for treating asthma.

4. A medicament for treating asthma, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The effective component comprises a fragment of a transport RNA molecule of Ginkgo medicinal herbs.

5. The medicament according to claim 4, characterized in that, The sequence of the fragment of the transport RNA molecule of the Ginkgo medicinal herbs comprises SEQ ID NO. 1 or a functional variant or homolog thereof.

6. The medicament according to claim 5, characterized in that, The RNA molecule is a double-stranded RNA molecule selected from any antisense sequence comprising SEQ ID NO. 1 or a functional variant or homolog thereof, and any complementary sense sequence comprising SEQ ID NO.

2.

7. The medicament according to claim 6, characterized in that, The double-stranded RNA molecule or a functional variant or homolog thereof, wherein the nucleotide sequence of the antisense strand of the double-stranded RNA molecule or a functional variant or homolog thereof is as shown in SEQ ID NO. 1, and the nucleotide sequence of the sense strand of the double-stranded RNA molecule or a functional variant or homolog thereof is as shown in SEQ ID NO.

2.

8. The medicament according to claim 6, characterized in that, The double-stranded RNA molecule or a functional variant or homolog thereof comprises a 3' overhang.

9. The medicament according to claim 6, characterized in that, The nucleotide sequence of the antisense strand and / or the sense strand of the double-stranded RNA molecule or a functional variant or homolog thereof comprises at least one chemically modified nucleotide; wherein the chemical modification is selected from at least one of 1-methyl, 2-methyl, 5-methyl, 7-methyl, N2-methyl, N6-methyl, N2,N2-dimethyl, 2'-O-methyl, N6-isopentenyl, 2-methylthio-N6-isopentenyl, N6-threonylcarbamoyl, N6-methyl-N6-threonylcarbamoyl, 2-thio, 4-thio, N4-acetyl, 5-formyl, 3-(3-amino-3-carboxypropyl), 5-methoxy, 5-oxoacetic acid, 5-oxoacetic acid methyl ester, 5-methoxycarbonylmethyl, 5,2'-O-dimethyl, 5-methoxycarbonylmethyl-2'-O-methyl, 5-methoxycarbonylmethyl-2-thio, 5-aminomethyl-2-thio, 5-methylaminomethyl, 5-methylaminomethyl-2-thio, 5-carbamoylmethyl, 5-carbamoylmethyl-2'-O-methyl, 5-carboxymethylaminomethyl, 5-carboxymethylaminomethyl-2'-O-methyl, 5-carboxymethylaminomethyl-2-methyl, 5-taurinomethyl, and 5-taurinomethyl-2-thio.

10. The medicament according to claim 4, characterized in that, The drug further comprises a nucleic acid stabilizer.

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