Application of SAL-0133 in preparation of medicine for preventing and / or treating human rhinovirus infection

Abstract

The invention provides application of SAL-0133 in preparation of a medicine for preventing and / or treating human rhinovirus infection, and relates to the technical field of medicines. It is found for the first time that the 3CLpro inhibitor SAL-0133 can effectively relieve and treat human rhinovirus infection, for example, the 3CLpro inhibitor SAL-0133 can significantly reduce lung virus titer after HRV-16 virus infection and significantly reduce overexpression of IL-6 and TNF-alpha inflammatory factor mRNA level. Indication of the 3CLpro inhibitor SAL-0133 is expanded, the 3CLpro inhibitor SAL-0133 can be further developed into a medicine for preventing and / or treating diseases caused by human rhinovirus infection, and a new way is provided for preventing and treating human rhinovirus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a use of SAL-0133 in preparing a medicament for preventing and / or treating human rhinovirus infection. Background Art

[0002] Human rhinovirus (HRV) belongs to the genus Enterovirus in the family Picornaviridae. It is a positive-sense, single-stranded RNA (ssRNA) virus with a genome length of approximately 7200 base pairs. The viral genome consists of a polyprotein encoded by a single open reading frame, which is cleaved by a viral protease to produce 11 proteins. VP1, VP2, VP3, and VP4 form the viral capsid that encapsidates the RNA genome, while the remaining non-structural proteins participate in viral genome replication and assembly. VP1, VP2, and VP3 determine the viral antigenic diversity, while VP4 mediates the anchoring of the RNA core to the capsid. A "canyon"-like structure on the surface of the VP1 protein serves as a key site for viral binding to cell surface receptors. HRV has the most serotypes in humans, with 120 types classified by serotype and antigenic typing, primarily into group A, group B, and group C rhinoviruses. Viral replication occurs through either endocytosis or pinocytosis, depending on the receptor type. Of the known HRV serotypes, over 90% utilize the cell surface receptor intercellular adhesion molecule-1 (ICAM-1) to enter cells, while a minority utilize the low-density lipoprotein receptor (LDLR) to attach to cells. Some antiviral drugs can induce a "canyon" shift, inhibiting viral adhesion and uncoating. Due to its lack of a lipid envelope, HRV is inactivated in organic solvents such as ether, chloroform, ethanol, and 5% benzene. HRV is heat-sensitive, losing infectivity at temperatures between 50°C and 56°C.

[0003] HRV can be transmitted between people through contact (direct contact or transmission via fomites) or aerosols (small or large particles). Intranasal or conjunctival inoculation can efficiently induce HRV infection, while oral routes cannot initiate the infection process. A large amount of evidence from experimental and clinical studies supports the role of HRV as a pathogen of the lower respiratory tract. Papadopoulos's team directly demonstrated that HRV can replicate efficiently in the lower airways through in vitro experiments on primary HBECs and in situ hybridization of bronchial biopsy specimens from experimentally infected healthy volunteers. After rhinovirus type 7 (RV-7) infection of HBECs, the expression levels of interleukin-6 (IL-6), IL-8, IL-16, and regulatory factor expressed and secreted by activated normal T cells (RANTES) were significantly increased compared with baseline. Gern et al. experimentally infected eight adult allergic volunteers with HRV-16. All subjects developed cold symptoms, and HRV-16 was cultured in nasal specimens. Two to four days after infection, HRV-16 was also detected by reverse transcription polymerase chain reaction (RT-PCR) in lower airway cells obtained via bronchoalveolar lavage (BAL). Experimental HRV infection in healthy adults also revealed transient abnormalities in lung function, such as frequency-dependent lung compliance.

[0004] In a few human histological reports of "HRV lower respiratory tract infection," HRV has been shown to induce both interstitial and alveolar lesions. Pathological findings in these studies include bronchiolitis obliterans with organizing pneumonia and interstitial pneumonia; acute and chronic inflammation with fibropurulent alveolar debris; and alveolar cell proliferation and desquamation. Together, experimental and clinical evidence confirm that HRV can infect the lower airways and induce a proinflammatory response. In addition to direct effects on respiratory epithelial cells, innate host responses also play a crucial role in the pathogenesis of human rhinovirus (HRV) infection. Triantafilou et al. conducted a series of in vitro experiments using primary human nasopharyngeal epithelial cells (HBECs) to investigate the specific mechanisms of HRV recognition by the innate immune system. They found that the HRV-6 capsid is recognized by TLR2; subsequently, after the HRV-6 single-stranded RNA (ssRNA) is internalized, the viral genome is recognized by TLR7 and TLR8 within the endosome. Once double-stranded RNA (dsRNA) is generated, a type I interferon (IFN) response is mediated by melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene 1 (RIG-1). Activation of these receptors maximizes HRV-induced expression of IFN-β and IFN-γ, as well as proinflammatory cytokine genes, including RANTES, IP-10, IL-6, IL-8, and epithelial cell-derived neutrophil-activating peptide 78 (ENA-78). Evaluation of cytokine gene promoter polymorphisms and their associated protein production may provide new insights into the manifestation of symptoms during HRV infection. In controlled studies of experimental and natural HRV infection, IL-6 levels were consistently elevated in nasal wash samples. This study found that subjects with low IL-6 expression (at position -174, C / C genotype) experienced more severe symptoms after experimental HRV-39 infection, despite no effect on nasal secretion production or mucociliary clearance time. The study also found that the IFN-γ (at position +874) phenotype predicted the frequency of seroconversion. IP-10 is a chemokine produced by bronchial epithelial cells, monocytes, lymphocytes, and neutrophils in response to IFN-γ and TNF-α cytokines, and HRV infection increases the levels of these cytokines. In nasal secretions from HRV-infected asthmatics and controls, the number of lymphocytes and neutrophils is significantly increased, accompanied by T cell infiltration of the airway epithelium and submucosa, and associated lymphopenia. Clearance elicits a stronger T cell response and subsequent cytokine release. Furthermore, T cell recruitment may contribute to the viral lytic process.

[0005] HRV infection can occur throughout the year and often forms epidemic peaks in autumn and spring (transmission is also active in winter in some areas). The epidemic characteristics in my country are regionally and seasonally heterogeneous. HRVA, B, and C types have been detected in respiratory infections, and the dominant serotype fluctuates with the year and region. As the primary pathogen of human respiratory infections, its clinical phenotype and severity are determined by the virus type, host immune status, and site of infection. It can cause upper respiratory tract symptoms such as the common cold, and can also induce or aggravate acute asthma attacks and acute exacerbations of chronic obstructive pulmonary disease (COPD). In infants, the elderly, and immunocompromised people, it can even progress to bronchitis and pneumonia. It is also associated with otitis media, sinusitis, etc. Human rhinovirus (HRV) infections are closely linked to ENT. Most infections are self-limited, but high-risk groups such as premature infants and immunocompromised individuals are prone to fatal outcomes such as respiratory failure, resulting in a significant disease burden. The development of anti-HRV drugs has long been hampered by the wide variety of serotypes and limited target specificity. Current key areas of focus include entry inhibitors, protease inhibitors (such as rupintrivir, which targets the 3C protease, but has not been approved due to local delivery limitations), host-directed therapies (interfering with host factors involved in viral replication requires a balance between efficacy and toxicity), and vaccines (traditional multivalent vaccines have limited feasibility, while novel strategies focusing on "pan-protection" are still in the preclinical stage). Currently, there are no approved antiviral therapies for the treatment of severe human rhinovirus infections. Certain DNA / RNA synthesis-inhibiting antiviral drugs (such as cidofovir, ganciclovir, and ribavirin) approved for the treatment of other viral infections have been clinically used for the treatment of severe human rhinovirus infections through expanded indications. However, in many cases, these drugs have limited efficacy and severe adverse reactions. Therefore, the development of other safer and more effective anti-HRV drugs is necessary.

[0006] SAL0133, a next-generation 3CLpro inhibitor, is a potential new therapeutic agent for the treatment of COVID-19. Preclinical data have shown significant anti-COVID-19 activity in vitro and in vivo. The project has completed preclinical studies and received approval for clinical trials from the China Development and Reform Commission (CDE) (CXHL2200945). It has essentially completed Phase I clinical trials, demonstrating a favorable safety profile, and is about to enter Phase II / III trials. However, no reports have been found regarding SAL-0133's potential use in the treatment of human rhinovirus infection. Summary of the Invention

[0007] In response to the above problems, the present invention provides a use of SAL-0133 in the preparation of a medicament for preventing and / or treating human rhinovirus infection.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a use of a 3CLpro inhibitor SAL-0133 in the preparation of a medicament for preventing and / or treating human rhinovirus infection.

[0009] Preferably, the structural formula of the 3CLpro inhibitor SAL-0133 is shown in Formula I below:

[0010] Formula I.

[0011] Preferably, the human rhinovirus comprises at least one of HRV-A, HRV-B and HRV-C.

[0012] Further preferably, the human rhinovirus is HRV-A.

[0013] Preferably, the human rhinovirus includes at least one of HRV-1, HRV-2, HRV-7, HRV-8, HRV-9, HRV-10, HRV-11, HRV-12, HRV-13, HRV-15 and HRV-16.

[0014] More preferably, the human rhinovirus is HRV-16.

[0015] Preferably, the drug inhibits the increase of lung viral load after HRV-16 infection.

[0016] Preferably, the drug inhibits the up-regulation of mRNA levels of inflammatory factors in the lungs after HRV-16 infection.

[0017] Preferably, the inflammatory factors include IL-6 and TNF-α.

[0018] Preferably, the active ingredient of the drug includes the 3CLpro inhibitor SAL-0133.

[0019] Further preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] Preferably, the auxiliary materials include but are not limited to excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavorings, fillers and antioxidants.

[0021] Preferably, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginic acid, gelatin, gum arabic, glyceryl monostearate, sodium starch glycolate, guar gum, glycerol, and propylene glycol.

[0022] Preferably, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0023] Preferably, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, and polyglyceryl-3 polyricinoleate.

[0024] Preferably, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0025] Preferably, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0026] Preferably, the binder is selected from at least one of ethanol, starch slurry, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinyl pyrrolidone, gum arabic, gelatin, and alginic acid.

[0027] Preferably, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0028] Preferably, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, and poloxamer.

[0029] Preferably, the pH adjuster is at least one selected from citric acid, fumaric acid, succinic acid, tartaric acid, malic acid and ascorbic acid.

[0030] Preferably, the flavoring agent is selected from at least one of sweet orange flavor, vanilla flavor, strawberry flavor, milk flavor, banana flavor, and cherry flavor.

[0031] Preferably, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0032] Preferably, the antioxidant can be selected from at least one of L-cysteine ​​hydrochloride, L-cysteine ​​base, 4,4 (2,3-dimethyltetramethylene dicatechol), tocopherol-rich extract (natural vitamin E), α-tocopherol (synthetic vitamin E), β-tocopherol, 6-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, octyl gallate, dodecyl gallate, tert-butylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid (vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate, ascorbyl palmitate, and ascorbyl stearate.

[0033] Preferably, the drug is administered orally.

[0034] Preferably, the dosage form of the drug is selected from any one of tablets, decoctions, powders, lozenges, capsules, granules, pills, and liquid preparations.

[0035] Compared with the prior art, the present invention has the following beneficial effects: (1) Currently, there are no approved antiviral drugs for human rhinovirus infection. The present invention has discovered a new anti-human rhinovirus drug, the 3CLpro inhibitor SAL-0133, which expands the indications of the 3CLpro inhibitor SAL-0133 and provides a new approach for the prevention and treatment of human rhinovirus.

[0036] (2) The present invention provides the use of the small molecule compound SAL-0133 in the preparation of a drug for inhibiting human rhinovirus and / or a drug for preventing and / or treating human rhinovirus infection, thereby providing a safe and effective small molecule compound for the clinical treatment of human rhinovirus. SAL-0133 can effectively inhibit the viral titer in the lungs of mice infected with human rhinovirus, alleviate pathology, and reduce the levels of inflammatory factors. It can be further developed into a drug for preventing or treating diseases caused by human rhinovirus infection and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Log is the viral load in the lungs of mice 8 hours after HRV-16 infection 10 TCID 50 ;in," " Compared with HRV-16 (model group), p <0.05.

[0038] Figure 2 is the level of inflammatory factors in the lungs of mice 8 hours after HRV-16 infection; ## " indicates that compared with the hICAM-1 mice in the blank group, p <0.01," ###" indicates that compared with the hICAM-1 mice in the blank group, p< 0.001," " Compared with HRV-16 (model group), p <0.05;" " Compared with HRV-16 (model group), p <0.01;.

[0039] Figure 3 This is the pathological image of the mouse lung 8 hours after HRV-16 infection. DETAILED DESCRIPTION

[0040] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific embodiment, but the following embodiment is only a preferred embodiment of the present invention, not all. Based on the embodiment in the embodiment, other embodiments obtained by those skilled in the art without making creative work all fall within the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their source is not specifically limited. The technology and scientific terms used in the embodiment have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0041] Example 1 Method for constructing an experimental model of hICAM-1 mice infected with HRV-16 virus S1. Twenty-four hICAM-1 mice aged 4-10 weeks were randomly selected and purchased from Guangdong Yaokang Biotechnology Co., Ltd. Both males and females were used.

[0042] S2. hICAM-1 mice were randomly divided into a normal group (no infection), an HRV-16 group (HRV-16 infected model group), a SAL-0133-H group (HRV-16 infected + SAL-0133 high-dose treated group), and a SAL-0133-L group (HRV-16 infected + SAL-0133 low-dose treated group), with 6 mice in each group.

[0043] Mice in the S3, SAL-0133-H, and SAL-0133-L groups were given oral administration of SAL-0133 twice daily starting 3 days before infection with HRV-16 virus at the following doses: SAL-0133-H: 500 mg / kg; SAL-0133-L: 250mg / kg.

[0044] S4. All mice were infected in a biosafety cabinet. After being anesthetized with isoflurane, 50 μL of HRV-16 virus was instilled into the nasal cavity. All other groups, except the control group, received PBS intranasally. Thirty minutes after infection, mice in the drug-treated group were gavage-administered SAL-0133.

[0045] S5. Wait for the mouse to recover from anesthesia and place it in a sterile cage to reduce possible cage contamination.

[0046] S6. The hICAM-1 mice of Example 1 were sacrificed 8 hours after infection with HRV-16, and lung tissues were obtained by dissection for subsequent experiments.

[0047] Example 2 Effect of SAL-0133 on HRV-16 virus-infected hICAM-1 mouse lung virus titer S1. The right lung tissue obtained by autopsy in Example 1 was homogenized and its viral titer was detected by cytopathic assay; S2. HeLa cells were seeded in a 96-well plate and cultured overnight. S3. Once cells have reached confluence, add 10-fold serial dilutions of mouse lung homogenate in DMEM (100 μL / well). Set up four replicate wells for each concentration. Also set up a normal control group and add an equal volume of DMEM as a blank control. Incubate at 37°C, saturated humidity, and 5% CO2. S4. After incubation for 4 hours, discard the supernatant, wash twice with PBS, and add basal culture medium to continue incubation for 72 hours.

[0048] S5. Observe cell growth daily. If cells show signs of cytopathic effect (CPE), such as shrinkage, rounding, or detachment, record the occurrence of CPE.

[0049] S6. Calculation of TCID in mouse lungs using the Reed-Muench method 50 value.

[0050] Test results such as Figure 1 It was shown that 8 hours after HRV-16 infection, oral administration of SAL-0133-H ( p <0.05) and SAL-0133-L ( p <0.05) The viral load in the lungs of the treatment group was significantly reduced.

[0051] Example 3 Effects of SAL-0133 on inflammatory factors in the lungs of hICAM-1 mice infected with HRV-16 The right lung tissue obtained by dissection in Example 1 was used to detect the expression of mouse lung inflammatory factors by qPCR.

[0052] S1. Lung homogenate was collected for RNA extraction; S2. Reverse transcription reaction The RNA was reverse transcribed into cDNA using the TaKaRa reverse transcription kit (Cat: RR036A) according to the reaction system in the kit instructions.

[0053] The cDNA obtained from the above reaction was used as a template, and the target gene primers (see Table 1) were added to detect the mRNA expression level of inflammatory factors in each sample using the q-PCR method (TaKaRa qPCR kit, Cat: RR390A).

[0054] The q-PCR reaction system (20 µL) is shown in Table 2: Table 1. Primer and probe sequences

[0055] Table 2. q-PCR reaction system

[0056] qRT-PCR reaction conditions are shown in Table 3: Table 3. qRT-PCR reaction conditions

[0057] The results are as follows Figure 2 As shown, compared with the hICAM-1 mice in the blank group, the expression of IL-6 and TNF-α inflammatory factors in HRV-16 (HRV-16 infected model group) mice was significantly different ( p <0.01)、( p <0.001), demonstrating that HRV-16 infection induced elevated levels of inflammatory factors in the lungs of hICAM-1 mice. Compared with the HRV-16 (HRV-16 infected model group), SAL-0133-H drug intervention treatment significantly reduced IL-6 and TNF-α gene expression levels ( p <0.05)、( p <0.01), and SAL-0133-L drug intervention treatment significantly reduced the TNF-α gene expression level ( p <0.01).

[0058] Example 4 Comparison of lung pathology results 8 hours after treatment in the HRV-16 group (HRV-16 infected model group), the blank group, and the SAL-0133-H and SAL-0133-L drug intervention groups. The left lung tissue obtained in Example 1 was stained with HE and observed under a microscope and photographed. Figure 3 No inflammatory cell infiltration and widening of alveolar septa were found in the lung tissues of mice in the blank group. In the HRV-16 infection group (HRV-16 infection model group), widening of the alveolar septa was observed, inflammatory cells mainly composed of neutrophils infiltrated in the alveolar cavity, and edema was observed around small blood vessels in the pulmonary interstitium. Compared with the HRV-16 infection group, the lung tissue lesions in the SAL-0133-H and SAL-0133-L groups were alleviated to varying degrees.

[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a 3CLpro inhibitor SAL-0133 in the preparation of a medicament for preventing and / or treating human rhinovirus infection.

2. The use according to claim 1, characterized in that The structural formula of the 3CLpro inhibitor SAL-0133 is shown in Formula I below: Formula I.

3. The use according to claim 1, characterized in that The human rhinovirus includes at least one of HRV-A, HRV-B and HRV-C.

4. The use according to claim 3, characterized in that The human rhinovirus is HRV-A.

5. The use according to claim 4, characterized in that The human rhinovirus includes at least one of HRV-1, HRV-2, HRV-7, HRV-8, HRV-9, HRV-10, HRV-11, HRV-12, HRV-13, HRV-15 and HRV-16.

6. The use according to claim 5, characterized in that The human rhinovirus is HRV-16.

7. The use according to claim 1, characterized in that The drug inhibits the increase in lung viral load after HRV-16 infection.

8. The use according to claim 1, characterized in that The drug inhibits the upregulation of mRNA expression of pulmonary inflammatory factors after HRV-16 infection.

9. The use according to claim 1, characterized in that The active ingredient of the drug includes the 3CLpro inhibitor SAL-0133, and the drug also includes pharmaceutically acceptable excipients.

10. The use according to claim 1, characterized in that The drug is administered orally; the dosage form of the drug is selected from any one of tablets, decoctions, powders, lozenges, capsules, granules, pills, and liquid preparations.

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