Use of raltitrexed in the manufacture of a medicament for the treatment of a disease caused by an adenovirus infection

Abstract

The present application provides the use of raltitrexed in the manufacture of a medicament for the treatment of diseases caused by adenovirus infection, including but not limited to respiratory tract infection, gastrointestinal infection and keratitis caused by adenovirus. When raltitrexed was tested for its anti-adenovirus activity at the cellular level, using a viral infection dose of 100 TCID50, the viral infectivity was determined on a human non-small cell lung cancer cell line (A549 cell) model, and the results showed that raltitrexed had a significant inhibitory effect on adenovirus, with an EC 50 = 8.98 nM, a CC 50 > 187.5 nM, and it was found that it had a significant inhibitory effect on the entry of the virus into the cell; and in the hDSG2 mouse model, a significant inhibitory effect of raltitrexed on viral replication was also observed. Therefore, raltitrexed can be used to treat diseases caused by adenovirus infection.

Description

[0001] This application claims priority to the following earlier application: Patent application No. 2024109680748, filed with the China National Intellectual Property Administration on July 18, 2024, entitled "Use of raltitrexed in the preparation of a medicament for treating diseases caused by adenovirus infection". The entire contents of this earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of raltitrexed in the treatment of adenovirus infections. More specifically, it relates to the use of raltitrexed and pharmaceutical compositions containing raltitrexed in the treatment of diseases caused by adenovirus infections, including but not limited to respiratory infections, gastrointestinal infections, and keratitis caused by adenoviruses. Background Technology

[0003] Human adenovirus (HAdV) belongs to the family Adenoviridae and the genus Adenovirus. It is a non-enveloped, double-stranded DNA virus with a genome length of approximately 33–36 kb. HAdV is an important pathogen causing respiratory infections and is widely distributed in nature. Besides infecting the respiratory tract, HAdV also exhibits broad tissue tropism, capable of infecting multiple sites including the digestive tract, conjunctiva, genitourinary tract, myocardium, and nervous system. To date, new HAdV variants have frequently emerged, with 113 variants identified, belonging to 7 subgenera (A–G).

[0004] Among them, HADV, which causes respiratory infections, mainly belongs to subgenuses B, C, and E, with the most common types being HADV-1–7, 14, 21, and 55. In my country, HADV-B3 and HADV-B7 are the main types prevalent in children, while HADV-B55 is an important pathogen causing community-acquired pneumonia in adults. Studies have shown that HADV-B55 is produced through homologous recombination of different HADV types, has a strong proliferative capacity, and has a high proportion of severe cases among infected patients.

[0005] In recent years, respiratory infections caused by adenoviruses have been prevalent in my country, and are highly likely to cause outbreaks. This not only affects the health of the general population but has also triggered multiple epidemics in local communities, seriously threatening public health security. If pneumonia caused by adenovirus infection is not treated promptly, the mortality rate in immunocompromised patients can be as high as 50%. Even in non-immunocompromised patients, the detection rate of adenovirus is rising. There are currently no specific drugs for treating adenovirus infection, either domestically or internationally. Currently, the adenovirus vaccines available in the United States mainly target types 4 and 7, but they do not have broad-spectrum efficacy.

[0006] Given the severity of adenovirus infection and the current lack of effective treatments, developing effective drugs against adenovirus infection is of great practical significance and urgency.

[0007] Raltitrexed (ZD-1694) is a quinolinyl folic acid analog that exerts its antitumor effect through the specific inhibition of thymidine synthase (TS). The drug is aggressively absorbed into cells via a reduced folic acid / methotrexate cell membrane carrier and rapidly and extensively metabolized intracellularly by folic acid polyglutamate synthase to its polyglutamate form. These polymeric forms of raltitrexed are more potent TS inhibitors than the original drug, and their inhibitory effect is more durable due to their prolonged intracellular retention. In preclinical studies, raltitrexed showed significantly stronger inhibitory effects on human colon cancer cell lines than the traditional antitumor drug fluorouracil.

[0008] Summary of the Invention

[0009] Although the inhibitory effect of raltitrexed on human colon cancer cell lines has been widely reported, its activity against adenovirus has not been reported. This invention tested the inhibitory activity of raltitrexed on adenovirus in an in vitro cell model, and the results showed that its half-maximal effective concentration (EC50) was [value missing]. 50 It reaches 8.98 nM, demonstrating a good inhibitory effect.

[0010] The technical solution of the present invention is as follows:

[0011] Use of raltitrexed in the preparation of medicines for treating diseases caused by adenovirus infection.

[0012] The treatment of diseases caused by adenovirus infection in this invention includes mild, moderate and severe infections caused by adenovirus, such as, but not limited to, respiratory infections, gastrointestinal infections and keratitis.

[0013] According to the present invention, the adenovirus has 113 types, belonging to 7 subgenera A to G.

[0014] According to the present invention, the adenovirus belongs to subgenus B, C, and E, and its types are HAdV-1 to 7, 14, 21, and 55.

[0015] According to the present invention, the adenovirus is HAdV-B3, HAdV-B7, or HAdV-B55.

[0016] The present invention also provides the use of raltitrexed in the preparation of pharmaceutical compositions for treating diseases caused by adenovirus infection.

[0017] According to the present invention, the pharmaceutical composition comprises raltitrexed and pharmaceutically acceptable excipients.

[0018] According to the present invention, the pharmaceutically acceptable excipient is selected from at least one of diluents, fillers, binders, disintegrants, lubricants, flow aids, surfactants, emulsifiers, solubilizers, coating materials, osmotic pressure regulators, preservatives, flavoring agents, antioxidants, colorants, and thickeners.

[0019] The present invention also provides a method for treating a disease caused by adenovirus infection, comprising administering raltitrexed to an individual in need of such treatment.

[0020] The raltitrexed of the present invention can be administered in unit dose form when treating diseases caused by adenovirus infection. The route of administration can be enteric or non-enteric, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, respiratory tract, skin, vagina and rectum.

[0021] The dosage forms for raltitrexed administration according to the present invention include, but are not limited to, oral liquids, tablets, capsules, injections, granules, powders, sustained-release formulations, and aerosols.

[0022] The raltitrexed of the present invention has the following beneficial effects in treating diseases caused by adenovirus infection:

[0023] In testing the anti-adenovirus activity of raltitrexed at the cellular level, a viral infection dose of 100 TCID50 was used in a human non-small cell lung cancer cell line (A549 cells) model to determine viral infectivity. The results showed that raltitrexed had a significant inhibitory effect on adenovirus, with an EC50 of [missing information]. 50 =8.98nM, CC 50 The viral load was >187.5 nM, and it was found to have a significant inhibitory effect after viral entry into cells. In animal studies, mice were infected with the viral stock solution, treated with raltitrexed only 2 days before infection. Lung tissue was collected 3 days after infection for viral load determination and pathological examination. Results showed that raltitrexed could inhibit viral replication in mice, and the pathological condition was improved. Therefore, raltitrexed can be used to treat diseases caused by adenovirus infection. Attached Figure Description

[0024] Figure 1 The dose-dependent changes in the inhibition of adenovirus infection of A549 cells by different concentrations of raltitrexed are shown, as well as the changes in the cytotoxicity of A549 cells by different concentrations of raltitrexed.

[0025] Figure 2A schematic diagram of the drug addition experiment described in Example 2 is shown. Wherein S1: Adenovirus infection without drug treatment; S2: Drug treatment at each stage; S3: Drug treatment before viral infection; S4: Drug treatment during viral incubation; S5: Drug treatment after viral infection.

[0026] Figure 3 The results show the viral DNA content after 40 hpi following the addition of 20 nM raltitrexed at different stages of viral infection (where **** indicates P < 0.0001 compared to the no-drug group).

[0027] Figure 4 The results showed that raltitrexed treatment significantly reduced viral expression in mouse lung tissue (where *** indicates P < 0.001 compared to the viral group).

[0028] Figure 5 This shows that raltitrexed treatment improved the pathological changes and inflammatory response in mouse lung tissue. Scale bar: 50 μm ( Figure 5 Image A shows lung tissue from the virus group; Image B shows lung tissue from the treatment group. Detailed Implementation

[0029] To illustrate the technical solution of the present invention, the following embodiments are provided, but the present invention is not limited thereto. The adenoviruses used in the following embodiments were all isolated from clinical strains, and raltitrexed (CAS: 112887-68-0) was purchased from Shanghai Taoshu Biotechnology Co., Ltd., with a purity ≥98%.

[0030] Example 1

[0031] Tests on dose-dependent changes of raltitrexed on adenovirus and its cytotoxic effects on A549 cells.

[0032] Test drug: Raltitrexed; 187.5 nM, 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, 10 nM, 5 nM, 1.25 nM.

[0033] Cells: A549 cells, purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in DMEM containing 10% FBS and 1% PS.

[0034] Virus: Adenovirus, titer 10 6 TCID 50 / ml, isolated from clinical strains and stored at -80℃. A viral titer of 100 TCID was used. 50 .

[0035] Sample preparation: Raltitrexed, purity ≥98%; prepared into a 100mM stock solution with DMSO and stored at -20℃ for later use.

[0036] Experimental method: Both the drug and the virus were diluted with DMEM containing 1% FBS and 1% PS.

[0037] (1) Add 100 μl of 5×10⁻⁶ solution to each well of a sterile 96-well culture plate. 4 A549 cells per ml were cultured at 37°C in a 5% CO2 incubator for 18-24 hours.

[0038] (2) The test drug was diluted to 9 concentrations, with 9 replicates for each concentration, 100 μl per well, and then an equal volume of 1% FBS and 1% PS DMEM was added to each well. The mixture was then incubated in an incubator for 2 h.

[0039] (3) After 2 hours, discard the mixture in the 96-well culture plate, and add 100 μl of the 9 concentrations of the test drug and 100 μl of 100 TCID to each well. 50 Virus solution, incubated in an incubator for 1 hour;

[0040] (4) After 1 hour, discard the mixture in the 96-well culture plate, wash with phosphate-buffered saline (PBS), and add 200 μl of DMEM containing 1% FBS and 1% PS to each well.

[0041] (5) Simultaneously set up cell control, blank control (solvent control) and virus control (negative control);

[0042] (6) The cells were incubated at 37°C in a 5% CO2 incubator for 40 hours;

[0043] (7) Observe cytopathic effects (CPE) under an optical microscope. Complete cytopathic effects are recorded as "++++", 75% cytopathic effects are recorded as "+++", 50% cytopathic effects are recorded as "++", 25% cytopathic effects are recorded as "+", and no cytopathic effects are recorded as "-".

[0044] (8) Extract viral DNA from each well cell and perform qPCR quantitative analysis. The inhibition rate is calculated using the formula: (virus control copy number - sample copy number) / virus control copy number * 100.

[0045] (9) Set up a drug-only treatment (gradient test), a cell control, and a blank control (cell-free) respectively. At 40 hpi, the supernatant was aspirated, and after washing with PBS, 100 μl of 10% CCK8 incomplete culture medium was added to each well. The cells were incubated at room temperature in the dark for 15 minutes, and the absorbance was measured at 450 nm. The cytotoxicity calculation formula is: (OD cell control - OD drug group) / (OD cell control - OD blank group).

[0046] Experimental conditions: All the above experimental operations were performed in the BSL-2 laboratory.

[0047] Result interpretation: The absence of CPE in cells indicates an effective concentration for virus inhibition, while the presence of CPE indicates ineffective inhibition.

[0048] Experimental results: using 100 TCID 50 In a human non-small cell lung cancer cell line (A549 cell model), the viral infection dose-dependent assay was performed using nine different doses of raltitrexed. The results are as follows: Figure 1 As shown.

[0049] Cell-level test results show that raltitrexed can inhibit adenovirus-induced cell damage, indicating that raltitrexed has in vitro anti-adenovirus activity.

[0050] Example 2

[0051] The drug addition experiment used qPCR to detect the inhibitory effect of raltitrexed on adenovirus infection at different dosing stages.

[0052] To further investigate the inhibitory effect of raltitrexed on adenovirus infection, this embodiment conducted a time-of-addition experiment (i.e., drug treatment at different stages of viral infection), and viral DNA was detected by qPCR.

[0053] Experimental Methods: A549 cells were seeded into 96-well plates and cultured for 18-24 hours until 85% coverage. Five groups were established. In groups S1, S4, and S5, the culture medium was replaced with 200 μl of DMEM containing 1% FBS and 1% PS. In groups S2 and S3, the culture medium was replaced with 200 μl of the drug diluted in DMEM containing 1% FBS and 1% PS, and incubated for 2 hours. After 2 hours, the culture medium in groups S1, S3, and S5 was replaced with 100 μl of DMEM containing 1% FBS and 1% PS; the culture medium in groups S2 and S4 was replaced with 100 μl of the corresponding drug concentration. Then, 100 μl of 100 TCID50 adenovirus was added to each group, and incubated for one hour. The culture medium in groups S1, S3, and S4 was replaced with 200 μl of DMEM containing 1% FBS and 1% PS, and the culture medium in groups S2 and S5 was replaced with 200 μl of the drug diluted in DMEM containing 1% FBS and 1% PS. The dosing schedule diagrams for each group are as follows: Figure 2 As shown. After incubation for 40 hours, DNA was extracted for qPCR. The qPCR results for each group are shown below. Figure 3 As shown.

[0054] Depend on Figure 3It is known that raltitrexed has a significant inhibitory effect on adenovirus after it enters the cell, effectively inhibiting the proliferation of viral progeny and reducing the production of infectious particles; this indicates that raltitrexed can treat diseases caused by adenovirus infection.

[0055] Example 3

[0056] Inhibitory effect of raltitrexed against adenovirus in mice

[0057] Female hDSG2 mice, aged 5-6 weeks, were used, with 5 mice in each group. There were two main groups: an observation group (group A: normal control, group B: raltitrexed group, group C: virus group, group D: raltitrexed treatment group); and an experimental group (group E: virus group, group F: raltitrexed treatment group). Mice in groups B, D, and F were intraperitoneally injected with 100 μl of raltitrexed only 2 days and 1 day before infection (-2 dpi, -1 dpi). On the day of infection (0 dpi), mice in groups C, D, E, and F were intranasally infected with 50 μl of virus. The observation groups were observed for one week: groups A and B showed no abnormalities; group C mice showed weight loss, decreased appetite, lethargy, and piloerection after infection; and group D mice showed reduced activity.

[0058] Three days post-infection (dpi), lung tissue was collected from the experimental group and divided into two aliquots. Viral DNA was extracted from one aliquot, and the other was placed in tissue fixation medium for pathological examination. Results were as follows: Figure 4 Figure 5 As shown. By Figure 4 The results showed that the relative expression level of viral DNA in the lung tissue of mice treated with raltitrexed was significantly lower than that in the untreated group, and the difference was statistically significant (P < 0.001). These results indicate that raltitrexed can inhibit viral replication in mice.

[0059] Figure 5 These are pathological images of lung tissue from mice in the virology and raltitrexed treatment groups. Figure 5 It can be seen that the pathological changes in lung tissue and the inflammatory response in mice treated with raltitrexed were significantly improved compared with those in the virus group.

[0060] In summary, raltitrexed has been shown to inhibit adenovirus infection in both in vivo and in vitro experiments, and therefore can be used to treat diseases caused by adenovirus infection.

[0061] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of raltitrexed in the preparation of a medicament for the treatment of diseases caused by adenovirus infection.

2. The use according to claim 1, characterized in that, Diseases caused by adenovirus infection include mild, moderate, or severe infections caused by adenovirus.

3. The use according to claim 1, characterized in that, Diseases caused by adenovirus infection include respiratory infections, gastrointestinal infections, and keratitis.

4. The use according to claim 1, characterized in that, The adenoviruses mentioned have 113 types, belonging to 7 subgenera from A to G.

5. The use according to claim 1, characterized in that, The adenovirus belongs to subgenus B, C, and E, and its types are HAdV-1 to 7, 14, 21, and 55.

6. The use according to claim 1, characterized in that, The adenovirus is HAdV-B3, HAdVB7, or HAdV-B55.

7. The use according to any one of claims 1-6, characterized in that, The drug is a pharmaceutical composition comprising raltitrexed and pharmaceutically acceptable excipients.

8. Use of raltitrexed in the preparation of pharmaceutical compositions for treating diseases caused by adenovirus infection.

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