Use of IACS-010759 in the preparation of a medicament for preventing and / or treating primary ciliary immobility syndrome

Abstract

The present invention provides a use of IACS-010759 in preparing a drug for preventing and / or treating primary immotile ciliary syndrome, belonging to the field of medicine. The present invention firstly finds that IACS-010759 can effectively restore the ciliary movement function, and has broad application prospects in preparing a drug for preventing and / or treating primary immotile ciliary syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the use of IACS-010759 in preparing a drug for preventing and / or treating primary ciliary immobility syndrome. Background Art

[0002] Primary ciliary dyskinesia (PCD) is a rare genetic disease, usually caused by defects in the structure or function of cilia. PCD is caused by gene mutations, which mainly affect the structure and function of cilia. Cilia are tiny, hair-like structures on the surface of cells. They play a key role in different types of cells, including the respiratory tract, middle ear and reproductive system. The disease can be inherited in an autosomal recessive manner, and there are also reports of X-chromosome inheritance, but it is relatively rare. According to foreign reports, the incidence of PCD is approximately 1:15000 to 1:20000, but due to the high rate of missed diagnosis and misdiagnosis, the actual incidence may be higher. The main clinical manifestations of PCD are: Chronic sinusitis: Due to abnormal ciliary function, mucus clearance is blocked, causing chronic inflammation, manifested as nasal congestion, runny nose, headache and other symptoms. Chronic bronchitis: Cilia dysfunction causes damage to respiratory epithelial cells, which are susceptible to infection and inflammatory reactions, manifested as repeated coughing and sputum. Immunodeficiency: Abnormal cilia structure may affect the migration and phagocytosis of white blood cells, reduce the body's ability to fight infection, and increase the risk of various infections. Situs inversus: About 50% of children have situs inversus, among which total situs inversus is manifested as right heart, two lobes of the right lung, three lobes of the left lung, and displacement of the spleen, liver, and stomach. Male infertility: Some male children may have fertility problems due to this disease.

[0003] To date, researchers have tried a variety of precision medicine approaches to treat primary ciliary dyskinesia (PCD), such as gene therapy, mRNA transcripts, and read-through therapy. Although gene therapy has shown potential in restoring ciliary activity, treatment still faces multiple challenges, including resistance of airway epithelium to apical transduction of viral vectors, achieving sufficiently high transfection rates to restore ciliary activity, inhibition of gene transfer by airway secretions, and difficulty in maintaining long-term efficacy. At the same time, fatal side effects that may be caused by immune responses and off-target effects must also be considered. Another downstream treatment method is transcription therapy or RNA therapy, which has the advantage of not changing genomic DNA and the effect is reversible. However, the effect of RNA therapy in the treatment of PCD is still unclear. In addition, the diffusion efficiency of mRNA encapsulated in lipid nanocarriers in lung tissue and the degradation of mRNA in cells still need to be further studied.

[0004] IACS-010759 is an orally available inhibitor of mitochondrial oxidative phosphorylation complex I (OXPHOS), a small molecule that inhibits ATP production in isolated mitochondria at low nanomolar levels. The metabolic effects of IACS-010759 are evident when malate and glutamate are administered but not when succinate is provided. In addition, the drug has favorable clinical properties with high oral bioavailability, excellent pharmacokinetic profile, and no adverse events. IACS-010759 is currently being evaluated in a clinical trial (NCT02882321; ClinicalTrials.Gov) to test its efficacy in patients with acute myeloid leukemia. There are no reports of IACS-010759 being used to treat primary immobile ciliary syndrome. Summary of the invention

[0005] The object of the present invention is to provide use of IACS-010759 in preparing a drug for preventing and / or treating primary ciliary immobility syndrome.

[0006] The present invention provides use of IACS-010759 in preparing a medicament for preventing and / or treating primary immotile cilia syndrome.

[0007] Furthermore, the drug is a drug that restores ciliary movement function.

[0008] Furthermore, the drug is a pharmaceutical preparation prepared with IACS-010759 as an active ingredient and pharmaceutically acceptable excipients.

[0009] Furthermore, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0010] Furthermore, the oral preparation is a tablet, a granule, or a capsule.

[0011] Furthermore, the injection preparation is a powder injection.

[0012] The present invention discovers for the first time that IACS-010759 can effectively restore the ciliary movement function and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile ciliary syndrome.

[0013] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0014] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The idea of ​​exploring whether IACS-010759 can restore the beating of respiratory cilia is as follows: the tracheal tissue of the respiratory tract is removed and processed, cultured into pulmonary trachea-like trachea, and the beating frequency of the cilia is restored after treatment with IACS-010759.

[0016] Figure 2 The results of identification of Dnah9 knockout efficiency in Dnah9 KD organoids: (A) is an agarose gel electrophoresis image showing the genotyping results of Dnah9 KD organoids; B is RT-PCR detection of Dnah9 knockout efficiency in Dnah9 KD organoids.

[0017] Figure 3 (A) Immunofluorescence verification of Dnah9 knockdown in organoids (red: Dnah9, green: P63 (epithelial cell marker)); (B) Observation results of secretory cells in organoids of Dnah9 knockdown mice (red: KART5 (red), green: Muc5ac (goblet cell marker)); (C) Observation results of ciliary morphology and number in organoids of Dnah9 knockdown mice (red: KART5 (basal cell marker), green: Ace-Tubulin (ciliary cell marker)); (D) Observation results of secretory cells in organoids of Dnah9 knockdown mice (red: KART5 (red), green: CC-10 (secretory cell-Club cell marker)).

[0018] Figure 4 This is a drug screening experiment for Dnah9 knockdown tracheal organoids: (A) is a schematic diagram of drug treatment; (B) is the organoid plating situation; (C) is the measurement of the ciliary beat frequency of Dnah9 knockdown tracheal organoids after drug treatment. DETAILED DESCRIPTION

[0019] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0020] Example 1: Effect of IACS-010759 in the treatment of primary ciliary immobility syndrome

[0021] 1. Experimental methods

[0022] The present invention refers to the prior art (Dnah9 mutant mice and organoid models recapitulate the clinical features of patients with PCD and provide an excellent platform for drug screening. Cell death&disease, 13(6),559.) and the method described in the "Dnah9 KD mice information" section on the left column of page 561 to construct an in vitro model of PCD caused by intrinsic dynein arm defects - lung organoids of Dnah9KO mice. Based on the organoids, an in vitro model for the treatment of PCD was provided, and the effect of IACS-010759 in treating primary ciliary immobility syndrome was studied ( Figure 1 ).

[0023] The specific experimental steps are as follows:

[0024] 1.1 Primary organoid culture plating

[0025] (1) Prepare 2 tubes of 1 ml digestion solution for use (Protease E 0.25 mg / ml, Collagense I 400 U / ml, Y27632 10 μm, DNase 10 U / ml).

[0026] (2) Take the lung lobes and trachea of ​​the mouse and place them in PBS. Use ophthalmic scissors and tweezers to cut the lung and tracheal tissue into pieces with a diameter of about 2 mm.

[0027] (3) Transfer the tissue blocks to the digestion solution for digestion (place in a shaker at 37°C, 100 rpm, and digest for 60 min).

[0028] (4) Use a pipette to blow and pump until the tissue pieces can pass through the pipette tip without resistance. When a large number of suspended cells are seen in the digestion solution, digestion can be stopped.

[0029] (5) Add 300ul FBS to terminate digestion, filter the tracheal tissue through a 40um filter to obtain the filtrate. Pass the lung lobe tissue through a 40um filter, discard the filtrate, and use PBS to reversely elute the unfiltered portion on the filter.

[0030] (6) After washing, remove the supernatant and retain the precipitate. Determine the amount of residual liquid based on the number of wells and mix it with the precipitate to make a cell suspension.

[0031] (7) Mix the liquefied Matrigel with the suspension (24-well: 30ul Matrigel, 15ul Matrigel for 48-well, 6ul for 96-well, concentration greater than 80%). When paving the wells, make sure to mix the tissue and Matrigel evenly so that they are evenly distributed in the Matrigel. Once completed, place the wells in a 37°C incubator to solidify the Matrigel for 15 minutes.

[0032] (9) Then add culture medium (24-well: 500ul; 48-well: 200ul; 96-well: 100ul). Replace the culture medium after 4 days or when it turns yellow.

[0033] 1.2 Organoid passaging

[0034] (1) Use a pipette to scrape off the organoids and matrix gel at the bottom, aspirate the suspension into an EP tube, and centrifuge at 200G for 3 minutes.

[0035] (2) Remove the supernatant, add 1 ml of cell recovery medium, pipette and mix thoroughly, place on ice for 20 min, and centrifuge.

[0036] (3) Washing: remove the supernatant, add 1 ml of WASH (washing solution), pipette to mix, and centrifuge at 200G for 3 min.

[0037] (4) Digestion: remove the supernatant, add 1 ml of Tryple to the EP tube, pipette and mix, place in a 37°C incubator for digestion for 5-10 min (take out and pipette every 5 min to observe the digestion status), take out the EP tube, pipette and mix until there is no white precipitate (if digestion is incomplete, the digestion time can be appropriately extended), add an appropriate amount of WASH to terminate digestion, and centrifuge at 200G for 3 min.

[0038] (5) Wash twice: Pour away the supernatant, add 1 ml of WASH, pipette to mix, and centrifuge at 200G for 3 min.

[0039] (6) 100um filtration: coarse filtration, used for organoids that need to be cultured quickly.

[0040] (7) Spreading gel: remove the supernatant, add an appropriate amount of Matrigel gel (30ul / well), mix well by pipetting, spread on a 24-well plate (30ul / well), and place in a 37°C incubator for 15 min.

[0041] (8) Take out the culture plate, add an appropriate amount of culture medium (0.5 ml / well), and place it in a 37°C incubator.

[0042] 1.3 Organoids as a platform for drug screening

[0043] After the pulmonary tracheal organoids were digested into single cells, they were replated in a 48-well plate. The drug IACS-010759 was diluted into the culture medium at different concentrations and added to each well. The control group was replaced with PBS. Each group was repeated 4 times and the drug treatment lasted for one week.

[0044] 1.4 Assessment of ciliary beating

[0045] Organoids from wild-type and Dnah9 knockout (Dnah9 KO) mice, as well as drug-treated organoids, were placed under an upright optical microscope (Olympus, CKX41) (connected to a high-speed camera (Optronis GmbH, Sprinter) and observed and photographed using a 100× oil immersion lens. The images were taken at a speed of 200 frames per second for 10 seconds and then converted to 0.1x for analysis.

[0046] 2. Experimental results

[0047] (1) PCD model verification

[0048] The present invention established a Dnah9 knockdown organoid. Genotyping was performed using agarose gel electrophoresis after PCR, proving that a homozygous knockout organoid had been obtained ( Figure 2 A). RT-PC results showed that Dnah9 was knocked out ( Figure 2 B).

[0049] (2) Establishment of Dnah9 KD lung tracheal organoids

[0050] The present invention then established Dnah9 knockdown organoids, and further confirmed the knockdown of Dnah9 protein levels by immunofluorescence staining ( Figure 3 A). By staining ciliated cells, the present invention observed that in Dnah9 knockdown organoids, the protein level of Muc5ac, a mucin produced by goblet cells of the respiratory mucosa, was significantly increased, which is consistent with the clinical manifestations of PCD patients ( Figure 3 B). In organoids with Dnah9 knockdown, the number of cilia decreased and their length became shorter ( Figure 3 C), while the number and morphology of secretory cells and goblet cells did not change ( Figure 3 D). This indicates that the PCD model was successfully built.

[0051] (3) Exploring the effect of IACS-010759 in treating PCD

[0052] The present invention uses Dnah9 knockdown organoids as a drug screening platform, uses NADH and ATP as positive controls (research has shown that these two small molecules can increase the frequency of ciliary beating, 10µM), sets three concentration gradients of IACS-010759 (5, 10, 50µM), adds Dnah9 knockdown organoids respectively, makes 4 sub-wells for each gradient, and continuously adds drugs for 16 days, after which the ciliary beating is measured ( Figure 4 A, B). Figure 4 B shows that compared with the Dnah9 knockdown organoid group without drug addition, the addition of IACS-010759, NADH or ATP all helped to restore the ciliary beating function. The results showed that 5µM or 10µM IACS-010759 was more effective than 10µM ATP and NADH drugs, and the ciliary beating frequency of 5µM IACS-010759 was significantly higher ( Figure 4 C). Therefore, the results showed that IACS-010759 has a significant effect in restoring ciliary motility.

[0053] The above results show that IACS-010759 has a significant effect on restoring ciliary motility and can be used to effectively treat primary ciliary immobility syndrome.

[0054] In summary, the present invention discovered for the first time that IACS-010759 can effectively restore ciliary motility and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile ciliary syndrome.

Claims

1. Use of IACS-010759 in the preparation of a medicament for preventing and / or treating primary immotile ciliary syndrome.

2. The use according to claim 1, It is characterized in that The drug is a pharmaceutical preparation prepared by using IACS-010759 as an active ingredient and adding pharmaceutically acceptable excipients.

3. The use according to claim 2, It is characterized in that The pharmaceutical preparation is an oral preparation or an injection preparation.

4. The use according to claim 3, It is characterized in that The oral preparations are tablets, granules and capsules.

5. The use according to claim 3, It is characterized in that The injection preparation is a powder injection.

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