Use of himabrodil in the preparation of a medicine against rabbit hemorrhagic disease virus

Abstract

The application discloses application of tamibarotene in preparation of a medicine for resisting rabbit hemorrhagic disease virus, and relates to the technical field of antiviral medicines. Through in silico knockout analysis of network pharmacology and in-vivo verification, it is found for the first time that tamibarotene can inhibit virus replication by targeting a host lipid metabolism pathway, thereby playing an excellent role in resisting RHDV2, and having the characteristics of oral effectiveness, a wide treatment window, high safety, and a high drug resistance barrier. After infection, administration of the tamibarotene can still significantly prolong the survival time, reduce the tissue virus load, and reduce pathological damage.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, specifically to the use of tamibarbital in the preparation of drugs against Rabbit Hemorrhagic Disease Virus type 2 (RHDV2, also known as GI.2). Background Technology

[0002] Rabbit Hemorrhagic Disease (RHD) is an acute, highly contagious, and fatal infectious disease caused by the rabbit hemorrhagic disease virus. It mainly infects domestic and wild rabbits and is characterized by hemorrhage and necrosis of the entire body's solid organs. The morbidity and mortality rates can reach over 90%. It is listed as a notifiable animal disease by the World Organisation for Animal Health (WOAH), and my country classifies it as a Class II animal disease.

[0003] Rabbit hemorrhagic disease virus (RHDV) belongs to the genus Lagovirus of the family Caliciviridae, and has two genotypes: classical RHDV (GI.1) and RHDV2 (GI.2). Compared with classical RHDV, RHDV2 has the following characteristics: a wider host range, infecting wild rabbits and various other rabbit species; it can infect young rabbits (under 35 days old), while classical RHDV mainly infects rabbits older than 2 months; existing classical RHDV vaccines do not provide sufficient cross-protection against RHDV2; and its mortality rate is as high as 70-90%.

[0004] Currently, prevention and control of RHDV2 still relies on vaccination. However, RHDV2 mutates rapidly, resulting in insufficient cross-protection from existing vaccines. Furthermore, there are currently no approved RHDV-specific antiviral drugs. Although broad-spectrum antiviral drugs such as ribavirin and interferon have been used in clinical trials, their efficacy is uncertain and they have significant side effects. Therefore, once infected with RHDV2, its high mortality rate can cause huge economic losses to the rabbit farming industry. Summary of the Invention

[0005] The purpose of this invention is to address the lack of specific therapeutic drugs for RHDV2 in the existing technology, and to provide the application of tamibarotene in the preparation of drugs against rabbit hemorrhagic disease virus. Tamibarotene (code name Am80) is a synthetically produced third-generation selective retinoic acid receptor α / β (RARα / β) agonist, chemically named 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid, with a molecular weight of 351.44. This drug inhibits RHDV2 viral replication by targeting the host's lipid metabolism pathway. It is characterized by oral efficacy, a wide therapeutic window, high safety, and a high resistance barrier. Post-infection administration can still significantly prolong survival time, reduce tissue viral load, and alleviate pathological damage.

[0006] The technical solution adopted in this invention is as follows:

[0007] The use of tamibarbitine in the preparation of drugs against rabbit hemorrhagic virus, wherein tamibarbitine is one or more of tamibarbitine raw material or its pharmaceutically acceptable salt, ester, or prodrug.

[0008] In this invention, tamibarbitine exerts its anti-rabbit hemorrhagic disease virus effect by inhibiting the SREBP-mediated lipid metabolism pathway, blocking PI4KA / OSBP / VAPA lipid transport, and inhibiting the formation of viral replication organelles.

[0009] Preferably, the rabbit hemorrhagic disease virus is rabbit hemorrhagic disease virus type 2.

[0010] Preferably, the dosage of tamilbarbitine is 25-75 mg / kg daily.

[0011] Preferably, the tamibarbitaline is administered to rabbits within 0-24 hours after infection with rabbit hemorrhagic disease virus, once daily for 3-7 consecutive days.

[0012] Preferably, the tamobarbitine is suspended in a 0.5% sodium carboxymethyl cellulose solution to prepare an oral formulation; the oral formulation is one of a suspension, tablet, capsule or granule.

[0013] The beneficial effects of this invention are as follows:

[0014] 1. Significantly prolonged survival time: Six hours after infection with RHDV2, New Zealand rabbits were administered 50 mg / kg tamibarbitine via gavage. The mean survival time increased from 11.0±0.8 hours in the solvent control group to 39.9±15.8 hours, a 3.6-fold increase (p<0.001); the mean survival time in the 75 mg / kg group was 40.3±6.0 hours, a 3.7-fold increase (p<0.001).

[0015] 2. Effectively reduces viral load in tissues: The viral load in the liver of the 50 mg / kg and 75 mg / kg groups decreased by about 0.5-1.5 log10 copies / mg compared with the control group, and the viral load in the heart, lungs and spleen decreased to varying degrees; the viral load in the kidneys of the 25 mg / kg group decreased by 1.5 log10.

[0016] 3. Significantly reduced pathological damage: In the treatment group, liver necrosis, inflammatory cell infiltration, and ballooning degeneration were significantly reduced; alveolar wall thickening and inflammatory cell infiltration in the lungs were reduced; and pathological scores of the spleen and kidneys decreased in a dose-dependent manner.

[0017] 4. No obvious cardiotoxicity: The cardiac pathology score of each dose group was 0, the myocardial structure was normal, and there was no bleeding, necrosis or inflammatory infiltration, which confirmed that the drug was safe. Attached Figure Description

[0018] Figure 1 It is a computer-simulated knockout (ISK) score ranking of the Top 10 RHDV2 host-dependent factors in an extended 215-node PPI network (screened from 210 host proteins).

[0019] Figure 2 shows the robustness analysis of the extended 215-node PPI network.

[0020] Figure 3 shows the distribution of the preferred host factors at each stage of the RHDV2 lifecycle.

[0021] Figure 4 shows the core factors of the extended RHDV2-host PPI network interacting directly with the virus.

[0022] Figure 5 shows the virtual mechanism of action of tamilbarbitine against RHDV2.

[0023] Figure 6 shows the results of in vivo pharmacodynamic experiments; Kaplan-Meier survival curves (n=3-12 / group).

[0024] Figure 7 shows the qPCR results of viral load in various tissues.

[0025] Figure 8 shows the pathological scores of each tissue. The gray dashed line indicates the time point for drug administration 6 hours after infection. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] 1. Experimental Materials

[0029] Drug preparation: Accurately weigh the tamibarbitine raw material (purity ≥99%), add 0.5% CMC-Na aqueous solution, grind to prepare a uniform suspension with concentrations of 2.5 mg / mL, 5.0 mg / mL and 7.5 mg / mL, respectively, store at 4℃ for later use, and sonicate before administration.

[0030] Laboratory animals: Healthy New Zealand White rabbits aged 35-40 days, weighing 1.5-2.0 kg, half male and half female, tested negative for RHDV antigen and antibody, and purchased from qualified laboratory animal farms. The experiment was approved by the Animal Ethics Committee of Sichuan Agricultural University (Approval No.: 20260312).

[0031] Virus strain: RHDV2 isolate (GenBank MT383749.1), passaged and rejuvenated in rabbits, used after determining LD50=100.

[0032] 2. Grouping and Dosing

[0033] Thirty-four New Zealand rabbits were randomly divided into five groups, as shown in Table 1.

[0034] Table 1

[0035]

[0036] Observe continuously for 7 days and record the time of death; perform immediate necropsy on animals that are near death or dead, and collect tissues from the heart, liver, spleen, lungs, and kidneys. One sample is used for viral load qPCR detection, and the other is fixed with 4% paraformaldehyde for HE staining and pathological scoring.

[0037] 3. Results Analysis

[0038] 3.1 The results of the survival analysis are shown in Table 2 and Figure 6 As shown.

[0039] Table 2

[0040]

[0041] Note: Compared with the control group, **p<0.01, ***p<0.001 (Log-rank test). There was no statistically significant difference between the 50 mg / kg and 75 mg / kg groups (p>0.05), suggesting that 50 mg / kg has reached the plateau of maximum effect.

[0042] 3.2 The RHDV2 VP60 gene copy number in each tissue was detected by qPCR. The result was standardized using the ΔCT value of the same plate control. The control group was set at 8.0 ± 0.3 log10 copies / mg. The viral load results are shown in Table 3. Figure 7 As shown.

[0043] Table 3

[0044]

[0045] The results showed that the viral load in the heart was lower in all tamibarbitaline dose groups than in the control group (0.72-0.88 times that of the control group); the viral load in the liver decreased in a dose-dependent manner, with the 75 mg / kg group decreasing to 0.38 times that of the control group (a 62% decrease); the viral load in the lungs also showed a decreasing trend (0.71-0.87 times); the 25 mg / kg group showed the most significant decrease in viral load in the kidneys (0.36 times, a 64% decrease). Individual differences existed in the viral load in the spleen and kidneys in the high-dose groups, but overall, the results suggest that the drug can effectively inhibit viral replication in multiple tissues.

[0046] 3.3 The results of pathological and histological examination are shown in Table 4 and Figure 8 As shown, HE staining pathological scores (0-4 points, 0 = normal, 4 = severe lesion).

[0047] Table 4

[0048]

[0049] No obvious pathological damage was observed in the heart in any group, confirming no cardiotoxicity. Pathological damage to the liver and lungs decreased in a dose-dependent manner: in the control group, extensive hepatocyte necrosis, hemorrhage, and inflammatory cell infiltration were observed in the liver; in the 50 mg / kg and 75 mg / kg groups, the liver structure was basically preserved, with only a small amount of punctate necrosis and inflammatory cell infiltration.

[0050] 3.4 Cell safety verification (CCK-8 assay)

[0051] The rabbit kidney cell (RK-13) CCK-8 assay showed that tamibarbitine had no significant inhibitory effect on cell viability in the concentration range of 0.1-100 μM; except for the extremely high concentration of 1000 μM, the other concentrations did not affect cell survival and even showed a tendency to promote cell proliferation, confirming that the drug has good safety and a wide therapeutic window.

[0052] This invention, through network pharmacology in silico knockout analysis combined with the above-mentioned in vivo administration experiments, first discovered that tamibarbitine exerts its anti-RHDV2 effect through the following mechanism (e.g. Figures 1-5 As shown in the diagram, it acts as a RARα / β agonist, activating the RAR signaling pathway; downstream, it inhibits the maturation and nuclear translocation of SREBP (sterol regulatory element-binding protein); downregulates the expression and function of lipid transport-related proteins such as PI4KA (phosphatidylinositol 4-kinase IIIα), OSBP (oxysterol-binding protein), and VAPA (VAMP-associated protein A); blocks the accumulation of cholesterol and phosphatidylinositol-4-phosphate (PI4P) required for viral replication on the membranes of replicating organelles; and inhibits the assembly and function of the viral replication complex, thereby blocking viral RNA replication and progeny virus assembly. This mechanism is a host-targeting antiviral strategy, not directly acting on viral proteins, thus possessing the advantage of a high resistance barrier.

[0053] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. The application of tamibarbitine in the preparation of drugs against rabbit hemorrhagic disease virus, characterized in that, The tamibarbitine is one or more of the tamibarbitine active pharmaceutical ingredient or its pharmaceutically acceptable salt, ester, or prodrug.

2. The application according to claim 1, characterized in that, The rabbit hemorrhagic disease virus mentioned is rabbit hemorrhagic disease virus type 2.

3. The application according to claim 1, characterized in that, The recommended dosage of tamilbarbitine is 25-75 mg / kg daily.

4. The application according to claim 1, characterized in that, The tamilbarbitine is administered to rabbits within 0-24 hours after infection with rabbit hemorrhagic disease virus, once daily for 3-7 consecutive days.

5. The application according to claim 1, characterized in that, The tamibarbitine is suspended in a 0.5% sodium carboxymethyl cellulose solution to prepare an oral formulation; the oral formulation is one of a suspension, tablet, capsule or granule.

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