Application of artemisinin and derivatives thereof in preparation of anti-adenovirus infection products

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of artemisinin and derivatives thereof in preparation of an anti-adenovirus infection product. It is found for the first time that artemisinin or derivatives thereof can be used for treating adenovirus infection. Artemisinin has HAdV activity, and the activity is irrelevant to a cell line through detection; the artemisinin inhibits HAdV infection by limiting virus replication in infected cells. The artemisinin derivative shows different degrees of cytotoxic cells and anti-HAdV activity to respiratory epithelial cells, with artemisinin as a reference, artesunate shows relatively high cytotoxicity, but the anti-HAdV activity is low, and artemisinone shows relatively low cytotoxicity and higher anti-HAdV activity. On a primary airway epithelial cell model, artemisinin and artemisinone serving as a derivative of artemisinin can inhibit HAdV infection, and the inhibiting effect is effective to different HAdV strains.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of artemisinin and its derivatives in the preparation of anti-adenovirus infection products. Background Art

[0002] Human adenovirus (HAdV) is a non-enveloped double-stranded DNA virus with more than 100 types belonging to 7 different subtypes. It can cause a variety of clinical manifestations, involving multiple systems such as the respiratory system, digestive system, and nervous system. It has a greater impact on infants and young children [1-3], accounting for 3.8% to 16% of acute lower respiratory tract viral infections [4,5]. In recent years, the incidence of adenovirus infection in children has been on the rise, which has attracted widespread attention [2,4-6]. Studies have shown that HAdV is most closely related to severe pneumonia, accounting for 32.2% of severe pneumonia, and is the main cause of death in children under 5 years old [9,10]. Severe adenovirus pneumonia has an acute onset, severe condition, and many complications. The mortality rate is as high as 10%, and 14% to 60% of children are left with complications, including obliterative bronchiolitis (PIBO), unilateral transparent lung, bronchiectasis, etc. ( Figure 1 ), which has an adverse effect on children's growth and psychological development, and also imposes a heavy burden on families and society[11-13]. Some seriously ill children still suffer from serious complications such as respiratory failure, lung collapse, brain, gastrointestinal and renal failure after active treatment, which can easily lead to death[2,13]. Although adenovirus infection can bring a considerable medical burden, effective antiviral therapies against these pathogens are still difficult to find[14-16].

[0003] Adenovirus infection is diagnosed as wind-heat attacking the lungs in TCM. The etiology is often caused by exogenous wind-heat invading the lung defense system. A variety of Chinese medicines can be used for treatment

[17] . Chinese medicines have advantages such as low drug resistance and multi-target mechanisms of action. Therefore, they have unique advantages and broad development prospects in the treatment of adenovirus infection. Screening for safe and effective antiviral Chinese medicines is one of the hot topics in the development of antiviral drugs [8]. Therefore, research on anti-adenovirus Chinese medicines has important social significance and economic value.

[0004] At present, the clinical treatment of adenovirus infection mainly relies on supportive treatment, including antipyretic, antiasthmatic, expectorant, nebulization and other symptomatic treatments, hormone anti-inflammatory, intravenous human immunoglobulin and other means. There are no antiviral drugs and methods for HAdV. Clinical use is limited to the use of broad-spectrum antiviral drugs such as ribavirin and cidofovir, which are not effective and have large side effects [3,7]. This highlights the urgency of developing new treatment strategies. One promising approach is to explore "drug repurposing", using existing drugs for new therapeutic indications, which provides a cost-effective and rapid method for drug development [18-20]. The repurposing of approved HAdV drugs has also been tested recently, and some studies have shown promising preclinical results [21,22].

[0005] Recent studies have found that some Chinese patent medicines (such as Reduning Injection

[23] ), single Chinese herbal extracts (such as Isatis indigotica

[24] ), and effective Chinese herbal ingredients (such as Shikonin

[25] ) all have anti-adenovirus effects. For adenovirus infection, Chinese herbal medicine has the advantage of multiple targets acting in combination and is not prone to drug resistance, which has unique advantages and broad development prospects[8].

[0006] Artemisinin is a sesquiterpene lactone isolated from the sweet wormwood plant (Artemisia annua). In recent years, it has been used as an effective ingredient in traditional Chinese medicine for its strong antimalarial properties

[26] . It is a highly effective and safe antimalarial drug and has been used to treat malaria in pregnant women and children

[27] . However, there are still no studies on whether it has anti-adenovirus effects.

[0007] Over the years, many artemisinin derivatives have been created and tested to improve the performance of artemisinin in treating malaria and other diseases. In addition to its antimalarial effects, there is growing evidence that artemisinin and its derivatives have broad-spectrum antiviral properties against a variety of viral pathogens, including herpes simplex virus (HSV), hepatitis B virus (HBV), hepatitis C virus (HCV), HIV-1, and SARS-CoV2 (16-19), mainly through inhibiting the activation of cellular transcription factors, interfering with the viral replication cycle, inducing apoptosis, and preventing the virus from binding to host cells

[18] . However, the effects of artemisinin and its derivatives on adenovirus infection remain understudied. Summary of the invention

[0008] The purpose of the first aspect of the present invention is to provide the use of artemisinin or its derivatives in the preparation of products for treating adenovirus infection.

[0009] The second aspect of the present invention aims to provide a method.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides the use of artemisinin or its derivatives in the preparation of products for treating adenovirus infection.

[0012] Preferably, the artemisinin or its derivatives include artemisinin, artemisinone, artesunate or pharmaceutically acceptable salts thereof.

[0013] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0014] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0015] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0016] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0017] Preferably, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

[0018] Preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0019] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0020] Preferably, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

[0021] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0022] Preferably, the adenovirus comprises human adenovirus (HAdV).

[0023] Preferably, the adenovirus comprises a respiratory-associated adenovirus.

[0024] Preferably, the adenovirus includes at least one of adenovirus HAdV1, 2, 3, 4, 5, 7, 11, 14, 21, and 55. The present invention tests the use of artemisinin or its derivatives in the treatment of HAdV3 and HAdV7, proving that artemisinin or its derivatives have a certain universality for HAdV virus. According to the teachings of the prior art (for example: Lai Xiaoxue, Zou Yingxue. Research progress on the immunological pathogenesis of adenovirus pneumonia in children [J]. International Journal of Pediatrics, 2022, 49 (5): 343-347. DOI: 10.3760 / cma.j.issn.1673-4408.2022.05.013.), those skilled in the art can expect that artemisinin or its derivatives can also be used to treat other respiratory tract infection-related adenoviruses, such as HAdV1, 2, 3, 4, 5, 7, 11, 14, 21, and 55. The specific type of adenovirus should not be a limitation of the present invention.

[0025] Preferably, the product comprises a medicament.

[0026] Preferably, the product comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0027] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0028] Furthermore, for the convenience of medication, the active ingredient artemisinin or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0029] Preferably, the dosage form of the product includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0030] Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0031] Furthermore, the dosage forms for administration through the gastrointestinal tract include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0032] Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0033] Furthermore, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0034] Preferably, the product is administered to a mammal.

[0035] Preferably, the mammal comprises a human.

[0036] A second aspect of the present invention provides a method for inhibiting adenovirus in vitro for non-therapeutic purposes, comprising the following steps:

[0037] The cells are treated with artemisinin or its derivatives.

[0038] Preferably, the artemisinin or its derivatives are screened in a corresponding concentration range according to a specific cell experimental system.

[0039] Preferably, the adenovirus comprises at least one of HAdV3 and HAdV7.

[0040] The beneficial effects of the present invention are:

[0041] The present invention innovatively screens out highly effective and low-toxic artemisinin and its derivatives as anti-adenovirus drugs in view of the current lack of specific anti-antiviral drugs. This is the first application of artemisinin and its derivatives in anti-adenovirus effects. It has opened up a new direction for the treatment of adenovirus infection-related diseases in the future. The details are as follows:

[0042] 1. Artemisinin has HAdV activity, and this activity has been tested to be independent of cell lines.

[0043] 2. Artemisinins inhibit HAdV infection by limiting viral replication in infected cells.

[0044] 3. Artemisinin derivatives showed different degrees of cytotoxicity and anti-HAdV activity against respiratory epithelial cells, with artemisinin as the reference (CC 50 =404.50±2.22μM; IC 50 =13.56±1.92μM), artesunate showed higher cytotoxicity (CC 50 =71.20±13.34μM), but the anti-HAdV activity was low (IC 50 =20.33 4.77 μM), while artemisinin showed lower cytotoxicity (CC 50 =659.29±34.56 μM) and higher anti-HAdV activity (IC 50 =5.91±0.80μM). This indicates that artemisinin derivatives have different anti-HAdV activity levels. Among the three tested derivatives, artesunate is less active than artemisinin, and artemisinone is more effective than artemisinin.

[0045] 4. In the primary airway epithelial cell model, artemisinin and its derivative artemisinin can inhibit HAdV infection, and this inhibitory effect is effective against different HAdV strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1The cytotoxicity and anti-human adenovirus (HAdV) activity of artemisinin were evaluated, wherein A represents that artemisinin was diluted in series and incubated with A549 and BEAS-2B cells for 72 hours, and then the cytotoxicity was measured by LDH detection, and the CC50 value was calculated; B represents that under the condition of adding different concentrations of artemisinin to the culture system, A549 and BEAS-2B cells were infected with HAdV3 virus for 24 hours, and then the virus infection level was detected and the IC50 value was calculated; the data shown are the mean ± standard deviation of 3 independent experiments.

[0047] Figure 2 The effects of artemisinin on HAdV binding to target cells, internalization, viral genome entry into the nucleus and viral replication; A means A549 cells were incubated with HAdV3 virus for 1 hour at 4°C with or without artemisinin, and then the unbound virus was washed and the level of virus bound to the cells was detected by PCR; B means A549 cells were incubated with HAdV3 virus for 1 hour at 37°C with or without artemisinin, and then the virus on the cell surface was removed with 0.25% trypsin-EDTA, and the internalized virus was detected by PCR; C means A549 cells were incubated with HAdV3 virus for 1 hour at 37°C with or without artemisinin, and then the virus on the cell surface was removed with 0.25% trypsin-EDTA, and the internalized virus was detected by PCR; A549 cells were incubated with HAdV3 virus for 1 hour with or without the addition of artemisinin, and then the cell nuclei were collected and the viral DNA in the nuclei was detected by PCR; D represents that A549 cells were incubated with HAdV3 virus for 24-48 hours at 37°C with or without the addition of artemisinin, and then the cells were collected and the viral DNA was detected by PCR; the data shown are the means ± standard deviations of three independent experiments; the solvent control group contained the same concentration of DMSO as the artemisinin (50 μM) group; ns, no statistically significant difference; *, p<0.05; **, p<0.01.

[0048] Figure 3 The present invention is to evaluate the cytotoxicity and anti-human adenovirus (HAdV) activity of artemisinin and its derivatives; wherein A indicates that artemisinin and its derivatives were serially diluted and incubated with A549 cells for 72 hours, and then the cytotoxicity was measured by LDH detection; B indicates that A549 cells were infected with HAdV3 virus for 24 hours in the presence of serially diluted artemisinin and its derivatives, and then the virus infection level was detected; the data shown are the mean ± standard deviation of 3 independent experiments; C indicates the calculation of CC50, IC50 and selectivity index (SI).

[0049] Figure 4 To evaluate the anti-human adenovirus (HAdV) activity of artemisinin and its derivatives; human epithelial cells from donor 1 (A) and donor 2 (B) were incubated with HAdV3 or 7 viruses for 24 hours with the addition of artemisinin or its derivatives, and then the level of viral infection was detected and the viral inhibition rate was calculated.

[0050] Figure 5 To evaluate the anti-human adenovirus (HAdV) activity of artemisinin; A549 and BEAS-2B cells were incubated with HAdV3 virus for 24 hours with or without the addition of gradient dilutions of artemisinin, and then the virus infection level was detected; the data shown are the means ± SD of 3 independent experiments.

[0051] Figure 6 Effects of artemisinin on the expression of human adenovirus early gene E1A; A549 cells were incubated with HAdV3 virus for 16 h at 37°C with or without the addition of artemisinin, and then the cells were collected and the viral DNA levels were detected by PCR; the data shown are the means ± SD of 3 independent experiments; the solvent control group contained the same concentration of DMSO as the artemisinin (50 μM) group.

[0052] Figure 7 Effect of artemisinin on the progeny virus titer of human adenovirus; A549 cells were incubated with HAdV3 virus for 48-72 hours at 37°C with or without the addition of artemisinin, and samples were then collected at 48 hours and 72 hours to measure the progeny virus titer; the data shown are the mean ± standard deviation of 3 independent experiments; the solvent control group contained the same concentration of DMSO as the artemisinin (50 μM) group.

[0053] Figure 8 To evaluate the anti-human adenovirus (HAdV) activity of artemisinin derivatives; A549 cells were incubated with HAdV3 virus for 24 hours with or without the addition of serial dilutions of artemisinin, and then the virus infection was detected. The data shown are the mean ± SD of 3 independent experiments. DETAILED DESCRIPTION

[0054] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0055] The relevant documents referred to in the present invention are as follows:

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[23] Feng Ganzhu, Zhou Feng, Huang Mao, et al. In vitro inhibitory effect of Reduning injection on adenovirus-3. Chinese Journal of New Drugs and Clinical Medicine, 2007, 26(8): 573-577

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[24] Deng Youping. Pharmacodynamic study on the anti-respiratory virus effect of Isatis indigofera extract. Wuhan: Wuhan University, 2013.

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[25] Gao H, Liu L, Qu ZY, et al. Anti-adenovirus activities of shikonin, component of Chinese herbal medicine in vitro. Biological&PharmaceuticalBulletin, 2011.34(2):197.

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[33] Marrugal-Lorenzo JA,Serna-Gallego A,González-González L, M, Poutou J, Pachón J, et al. Inhibition of adenovirus infection by mifepristone. Antiviral research. 2018; 159:77-83.

[0089] Example 1 Artemisinin has the activity of inhibiting HAdV

[0090] 1. Drugs, cells and viruses

[0091] Artemisinin (CAS No.: 63968-64-9; purity 99.83%) and artesunate (CAS No.: 88495-63-0; purity 99.56%) were purchased from Selleckchem, and artemisinin (CAS No.: 255730-18-8; purity ≥ 98.0%) was purchased from MedChemExpress. The compounds were first dissolved in DMSO at a concentration of 200 mM as a stock solution, aliquoted and stored at -20°C. The stock solution was then further diluted to the concentration required for the cell experiment.

[0092] All cell lines used in the present invention were obtained from the American Culture Collection (ATCC) and cultured according to the recommendations of the ATCC. Human lung epithelial cell line A549 and human cervical epithelial cell line HeLa were cultured in DMEM (GenomBio) supplemented with 10% FBS (Hyclone), and human lung epithelial cell line BEAS-2B were cultured in BEGMTM Bronchial Epithelial Growth Medium (Lonza). Primary human bronchial epithelial cells were purchased from PromoCell and cultured in Airway Epithelial Cell Growth Medium (PromoCell). All cell cultures were cultured in a 37°C incubator containing 5% carbon dioxide. Antibiotics (GenomBio) may be added as needed.

[0093] Two HAdV strains, HAdV 3 and HAdV 7, were used in this study, both from ATCC and propagated according to the recommendations of ATCC. A549 and HeLa cells were used for the propagation of HAdV3 and HadV 7, respectively. The cells first reached 70-85% confluence in a T175 flask and then inoculated with the virus at 0.1 MOI. After 3-4 days of culture, the virus was harvested and purified by two-step CsCl gradient centrifugation. The virus was first released from the infected cells by 3 freeze-thaw cycles and then centrifuged at 10,000g for 10 minutes to remove cell debris. The supernatant was then cleared and loaded onto 2.2 and 4M CsCl discontinuous gradients and centrifuged at 25,000rpm for 2 hours (SW28, Beckman Coulter). The virus band was harvested, mixed with saturated CsCl in a 1:1 ratio, and with another 2.2 and 4M CsCl discontinuous gradient, and then centrifuged at 35,000rpm for 3 hours (SW41Ti, BeckmanCoulter). All centrifugation was performed at 4°C. The virus buffer was exchanged into PBS + 10v / v% glycerol ultracentrifugation column (Merck) using Amicon purification and aliquoted and stored at -80°C. To check the virus titration, the stored virus aliquots were thawed at 37°C and then diluted 10 times in a complete cell culture medium. After dilution, the virus samples were then added to the target cell monolayer in a 96-well plate and cultured for 48 hours. Subsequently, the cells were washed with PBS and fixed with cold methanol. The number of HAdV-positive cells was then detected using the adenovirus DFA kit (EMD Millipore).

[0094] 2. Experimental methods

[0095] 1) Cytotoxicity assay

[0096] The cytotoxicity of artemisinin, artesunate, and artemesunone was assessed by LDH and MTT. A commercial LDH-Cytox assay kit (BioLegend) and a commercial MTT cell proliferation and cytotoxicity assay kit (Beyotime) were used according to the manufacturer's instructions.

[0097] For the LDH assay, artemisinin, artesunate and artemisinin were first serially diluted with complete cell culture medium and then added to cells pre-seeded in 96-well plates and cultured for 72 hours. After incubation, LDH working solution was added to each well, incubated for 30 minutes at room temperature in the dark, and then the stop solution was added. The plate was then read for absorbance at 490nm and cytotoxicity was calculated. In order to evaluate the effect of the solvent on cell viability, the highest DMSO concentration of the diluted drug was used as a blank negative control, and the lysis buffer (from the detection kit) was used as a positive control as a 100% comparison benchmark when calculating viability, as a comparison benchmark for 100% cell death.

[0098] For the MTT assay, cells were treated in the same manner as for LDH, and after 72 h of culture, 10 μL of MTT solution was added to each well and the plate was incubated at 37 °C for 4 h. After incubation, 100 μL of MTT solution was added to each well and further incubated at 37 °C until the formazan crystals were completely dissolved. The plate was then read at 570 nm to measure the absorbance and calculate cell viability. To evaluate the effect of the solvent on cell viability, the highest DMSO concentration of the diluted drug was used as a blank negative control and used as a 100% comparison benchmark when calculating viability.

[0099] 2) HAdV activity inhibition test

[0100] The virus inhibition test was performed with reference to relevant literature

[28] . Serially diluted artemisinin and its derivatives were first incubated with HAdV at 37°C for 1 hour, and then the mixture was added to A549 or BEAS-2B cells pre-seeded in 96-well plates and cultured for an additional 24 hours. Then, the cells were washed with PBS and treated with 100% cold methanol. The number of virus-infected cells was detected using the adenovirus DFA kit (EMDMillipore). The inhibition rate of artemisinin against the virus was expressed as IC50.

[0101] 3. Experimental results

[0102] Two human airway epithelial cell lines, A549 and BEAS-2B, were used to confirm that the inhibitory effect of artemisinins on adenovirus was not cell-dependent.

[0103] After 100 μM concentration, the results increased in a dose-dependent manner ( Figure 1 The results showed that artemisinin had no cytotoxic effect. The results of the concentration range of artemisinin without cytotoxicity are as follows Figure 1 C and Figure 5 As shown, artemisinin inhibited HAdV3 infection in a dose-dependent manner, and this inhibitory effect was observed at similar potency levels in both A549 and BEAS-2B cells.

[0104] Specifically, the IC of artemisinin in A549 cells 50 The concentration of artemisinin in BEAS-2B cells was 11.58±1.80 μM, while the concentration of artemisinin in BEAS-2B cells was 15.50±2.22 μM. In summary, the experimental data showed that artemisinin has the activity of inhibiting HAdV, and this activity has nothing to do with the cell line after detection.

[0105] Example 2 Artemisinin exerts anti-HAdV effect by inhibiting viral replication

[0106] 1. Experimental methods

[0107] 1) Virus binding assay

[0108] The virus binding test was performed with reference to the relevant literature

[28] . A549 cells were first incubated with HAdV (200 MOI) at 4°C for 1 hour, and then the cells were washed with cold sterile PBS to remove unbound viruses. After washing, the cells were harvested and total DNA was extracted using a commercial magnetic pathogen DNA kit (Vazyme). Then, with reference to the relevant literature [29,30], the viral DNA was quantitatively analyzed by real-time PCR (RT-PCR).

[0109] The relative titer of HAdV in the sample was determined by 2 -ΔΔ The Ct method was used for calculation, and GAPDH was used as an internal standardization control. The following primers were used for HAdV detection: nehex3deg (5'-GCCCGYGCMACNGANACSTACTTC-3', SEQ ID NO.1) and nehex4deg (5'-CCYACRGCCAGNGTRWANCGMRCYTTGTA-3', SEQ ID NO.2). These primers bind to the conserved regions of the hexon gene of different HAdV subtypes.

[0110] The following primers were used for GAPDH internal control: GAPDH-F: (5'-GAGTCAACGGATTTGGTCGT-3', SEQ ID NO. 3) and GAPDH-R (5'-TTGATTTTGGAGGGATCTCG-3', SEQ ID NO. 4). The PCR protocol was as follows: initial denaturation at 98°C for 3 minutes, followed by 40 cycles of 98°C, 30 seconds and 60°C, 30 seconds each.

[0111] 2) Virus internalization assay

[0112] The virus internalization assay followed a previously reported protocol with some modifications [28,31]. Briefly, A549 cells were first incubated with HAdV (in the presence or absence of artemisinin) at 37°C for 1 h to allow the virus to infect and enter the target cells (no virus-drug preincubation was required before cell treatment).

[0113] After incubation, cells were immediately transferred to ice to stop the reaction. After incubation, virus-containing medium was removed, cells were washed with PBS and dissociated by treating with 0.25 wt% trypsin-EDTA solution at 37°C for 5 minutes, followed by washing with PBS. Total DNA was then isolated from washed cells, and the quantification of HAdV DNA was detected using the same RT-PCR method as described for virus binding.

[0114] 3) Virus nuclear entry assay

[0115] The virus nuclear entry test was performed according to the relevant literature

[32] . First, the cells were incubated with adenovirus at 37°C for 1 hour with or without the addition of artemisinin, and then the virus that was not bound to the cell surface was removed using 0.25wt% trypsin-EDTA solution, and then washed with cold PBS. After washing, the nuclei were isolated from the precipitated cells using a commercial cytoplasm and nucleus isolation kit (ProteinTech), and the viral DNA was extracted using a commercial magnetic pathogen DNA kit (Vazyme), and the level of viral genome in the nucleus was detected by RT-PCR. All experiments were performed according to the manufacturer's instructions.

[0116] 4) Virus replication assay

[0117] Virus replication experiments were performed according to the relevant literature

[33] . First, cells were incubated with adenovirus at 37°C for 24-48 hours with or without the addition of artemisinin. Cells were then collected and DNA was extracted using a commercial DNA isolation kit (Vazyme) according to the manufacturer's instructions. The level of viral genome in the cell nucleus was detected by RT-PCR.

[0118] 5) Determination of progeny virus titer

[0119] A549 cells were infected with HAdV virus and cultured for 72 hours with or without the addition of artemisinin. Samples were collected at 48 and 72 hours, and the titer of progeny virus was determined using the adenovirus DFA kit (EMD Millipore).

[0120] 2. Experimental results

[0121] This example further studies artemisinin-induced HAdV inhibition. Successful HAdV infection can be divided into four steps: virus binding to target cells, virus internalization by target cells, viral genome entry into the nucleus of infected cells and viral replication in target cells. This example aims to examine which cycle step of HAdV-infected cells is affected by artemisinin. Because the activity of anti-artemisinin against HAdV does not depend on the cell line, subsequent experiments were performed only in A549 cells. First, the effect of artemisinin on the binding of the virus to target cells was studied. Figure 2 As shown in Figure A, 10 or 50 μM artemisinin did not cause significant changes in the number of cell-bound viruses, indicating that artemisinin did not affect viral attachment. Similarly, internalization assays showed that the number of viral genome copies in artemisinin-treated and untreated groups was comparable, indicating that the drug did not alter the process of viral entry into target cells ( Figure 2 Quantitative analysis of the nuclear-associated viral genome confirmed that artemisinin did not affect the nuclear entry of the HAdV3 genome, as the genome levels in treated and untreated cells were comparable ( Figure 2Finally, viral replication was assessed and it was found that HAdV replication was reduced in the presence of artemisinin in a dose- and treatment-time-dependent manner ( Figure 2 (D in the figure). The inhibitory effect of artemisinin on HAdV3 replication was stronger at 48 hours after infection than at 24 hours, indicating that viral replication at 24 hours was lower than that at 48 hours. This experiment also tested the effect of artemisinin on the expression of the adenovirus early gene E1A. The data showed that artemisinin treatment did not significantly change the E1A RNA level, indicating that artemisinin inhibits HAdV3 replication through a mechanism independent of E1A ( Figure 6 ).

[0122] In addition, this example evaluates whether artemisinin can also reduce the production of viral progeny by inhibiting viral replication. The data showed that the progeny viral load in the artemisinin-treated group was significantly lower than that in the untreated group, and the level of progeny viral reduction was related to the artemisinin dose and treatment time ( Figure 7 ). Taken together, the data suggest that artemisinins inhibit HAdV infection by limiting viral replication in target cells.

[0123] Example 3 Artemisinin derivatives exhibit different degrees of cytotoxicity and anti-HAdV activity on respiratory epithelial cells

[0124] This example compares the cytotoxicity and anti-HAdV activity of different artemisinin derivatives to confirm whether the artemisinin derivatives have anti-HAdV activity. The relevant experimental steps refer to Examples 1 and 2.

[0125] Two artemisinin derivatives, artesunate and artemesunone, were tested. The data showed that artemisinin and its two derivatives all showed anti-HAdV activity, but their cytotoxicity and anti-HAdV spectra were very different ( Figure 3 AD and Figure 8 Specifically, artemisinin was used as a reference (CC 50 =404.50±2.22μM; IC 50 =13.56±1.92μM), artesunate showed higher cytotoxicity (CC 50 =71.20±13.34μM), but the anti-HAdV activity was low (IC 50 =20.334.77 μM), while artemisinin showed lower cytotoxicity (CC50 = 659.29 ± 34.56 μM) and higher anti-HAdV activity (IC 50 =5.91±0.80 μM). Selectivity index (SI=CC 50 / IC 50) evaluated and calculated the safety and efficacy of these three anti-HAdV drugs. The data showed that artemisinin performed best among the three drugs, with SI = 111.55, followed by artemisinin (SI = 29.83), and finally artesunate (SI = 3.50; Figure 3 (middle D).

[0126] In summary, the data showed that artemisinin derivatives differed in their levels of activity against HAdV, and among the three derivatives tested, artesunate was less active than artemisinin, and artemisinone was more effective than artemisinin.

[0127] Example 4 Evaluation of the activity of artemisinin and its derivative artemisinin against HAdV using an airway epithelial cell model

[0128] In order to further confirm the relevant research results, this example next evaluates the effect of artemisinin and its derivatives on the human primary airway epithelial cell model. Since the efficacy of artesunate was not as good as artemisinin in previous tests. Therefore, this example only tested artemisinin and its derivative artesunate. In order to further confirm that the anti-HAdV activity of artemisinin and its derivatives is not only effective against HAdV 3, another common adenovirus type HAdV7 that causes respiratory infections was also introduced in this experiment. Please refer to Examples 1 and 2 for relevant experimental steps.

[0129] like Figure 4 As shown in A and B, both artemisinin and artemisinone inhibited HAdV infection in the primary respiratory epithelial cell model. Consistent with previous results, artemisinone still showed better antiviral activity than artemisinin. In addition, both drugs inhibited HAdV 7 infection to a level similar to that of HAdV 3, which means that their anti-HAdV activity is not restricted by HAdV strains ( Figure 4 (A, B).

[0130] In summary, the data of the present invention confirm that artemisinin and its derivative artemisinin can inhibit HAdV infection in a primary airway epithelial cell model, and this inhibitory effect is effective against different HAdV strains.

Claims

1. Use of artemisinin or its derivatives in the preparation of products for the treatment of adenovirus infection.

2. The use according to claim 1, characterized in that: The artemisinin or its derivatives include artemisinin, artemisinone, artesunate or pharmaceutically acceptable salts thereof.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

4. The use according to claim 1, characterized in that: The adenovirus includes a respiratory-associated adenovirus; Preferably, the adenovirus includes at least one of adenovirus HAdV1, 2, 3, 4, 5, 7, 11, 14, 21, and 55.

5. The use according to claim 1, characterized in that: The products include pharmaceuticals.

6. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 5, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. The use according to claim 8, characterized in that: The product is administered to a mammal; Preferably, the mammal comprises a human.

10. A method for inhibiting adenovirus in vitro for non-therapeutic purposes, comprising the following steps: The cells are treated with artemisinin or its derivatives.

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