Use of lapatinib in the preparation of a drug against rabies virus

Abstract

The application discloses application of lapatinib in preparation of a rabies virus resisting medicine, and first discovers the rabies virus resisting activity of lapatinib, breaks the limitation of lapatinib only being used for treating tumors, and first proves the role of lapatinib in inhibiting rabies virus replication through in-vivo and in-vitro experiments, develops the potential application value of lapatinib in early treatment after rabies virus exposure, and provides a new use of lapatinib, i.e., using lapatinib as a candidate medicine for rabies treatment, especially specific treatment in the early stage of rabies virus infection, and fills the blank of specific treatment medicines after rabies virus exposure.

Description

Technical Field

[0001] This invention relates to new applications of pharmaceuticals, and more particularly to the use of lapatinib in the preparation of anti-rabies virus drugs. Background Technology

[0002] Lapatinib, chemical name: N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methanesulfonylethylamino)methyl]-2-furanyl]-4-quinazolinamine, CAS number: 231277-92-2, is a synthetic small molecule inhibitor targeting the dual HER2 / EGFR tyrosine kinase. Its molecular structure contains a quinazolinoid core (competitive binding site with ATP) and a furan ring side chain (enhancing receptor binding specificity).

[0003] This compound was initially used for anti-tumor therapy and was approved by the US FDA in 2007 for the clinical treatment of HER2-positive advanced breast cancer (trade name: Tykerb). The specific indication was "in combination with capecitabine for the treatment of HER2-positive metastatic breast cancer that has failed prior anthracycline, taxane, and trastuzumab therapy" (refer to FDA approval document: NDA 022049). The mechanism of action is as follows: lapatinib competitively binds to the ATP-binding sites in the intracellular regions of the HER2 (ErbB-2) and EGFR (ErbB-1) receptors, inhibiting receptor autophosphorylation and activation of downstream signaling pathways (RAS-MAPK pathway, PI3K-AKT pathway), blocking tumor cell proliferation signal transduction and anti-apoptotic pathways, ultimately inhibiting tumor growth (refer to patent (US): US7358256B2).

[0004] The current clinical application characteristics are as follows:

[0005] (1) Dosage form: It is mainly made into oral film-coated tablets in the form of hydrochloride (lapatinib hydrochloride monohydrate, which has better stability than free base);

[0006] (2) Dosage: The usual dose is 1250 mg / day, in combination with capecitabine (1000 mg / m², twice daily), with each cycle lasting 21 days;

[0007] (3) Pharmacokinetics: The oral bioavailability is about 70% (absorption can be improved when taken with food), the half-life is 24 hours, it can cross the blood-brain barrier, and the cerebrospinal fluid concentration is 10%-20% of the plasma concentration (refer to Clinical Pharmacology & Therapeutics, Vol. 80, No. 3, 2006, pp. 258-269).

[0008] (4) Safety: Adverse reactions are mainly mild to moderate, including diarrhea (occurrence rate of about 65%, mostly grade 1-2) and rash (occurrence rate of about 40%). There is no serious central nervous system toxicity (refer to the adverse reaction section of the FDA drug instructions).

[0009] Lapatinib is currently only approved for the treatment of HER2-positive advanced breast cancer. Its clinical application and research are limited to the field of oncology. There are no literature or patent reports on its anti-rabies virus activity, nor has it been used for the prevention or treatment of rabies. Its potential in the field of antiviral therapy has not yet been explored.

[0010] Rabies is a highly fatal acute infectious disease of the central nervous system caused by the rabies virus (RABV). It is mainly transmitted through bites / scratches of infected animals. Globally, approximately 59,000 people die from rabies each year, with 95% of these deaths occurring in remote and impoverished areas such as Africa and Asia (WHO, 2023 Global Rabies Report).

[0011] The virus core is a single-stranded negative-sense RNA that encodes five proteins: nucleoprotein (N, which wraps the RNA to form a nucleocapsid), phosphoprotein (P, which helps viral replication), matrix protein (M, which mediates viral particle assembly), glycoprotein (G, which binds to host cell receptors), and RNA polymerase (L, which catalyzes viral RNA replication). The key steps of pathogenesis are: a) the virus invades the peripheral nerves by binding to nicotinic acetylcholine receptors at the nerve endings via the G protein; b) it is retrogradely transported along the axon to the central nervous system; c) it replicates extensively within neurons, damaging nerve cell function and causing acute encephalitis, with a mortality rate of almost 100% after onset.

[0012] Currently, rabies prevention and control mainly relies on pre-exposure prophylaxis (PEP) and post-exposure prophylaxis (Post-Exposure Prophylaxis). The specific protocols are as follows:

[0013] Pre-exposure vaccination:

[0014] High-risk occupational groups: veterinarians, animal handlers, rabies laboratory staff, and disease control personnel.

[0015] High-frequency contact groups: pet industry workers (grooming, training), people who frequently come into contact with wild animals, and long-distance travelers (to areas where rabies is prevalent).

[0016] Special needs groups: family members of animals with a high risk of rabies (such as unvaccinated dogs and cats) or individuals with weakened immune systems (protection needs to be established in advance).

[0017] Post-exposure prophylaxis:

[0018] Wound treatment: Immediately rinse the wound with soapy water (or 0.1% benzalkonium bromide) and running water alternately for more than 15 minutes, then disinfect with povidone-iodine.

[0019] Vaccination: Vero cell purified rabies vaccine is commonly used, and the whole course is carried out by the "5-dose regimen" (one dose each on days 0, 3, 7, 14 and 28) or the "2-1-1" four-dose regimen (two doses on day 0, and one dose each on days 7 and 21).

[0020] Immunoglobulin injection: For Category III exposures (penetrating skin bites, head and face exposures, and mucosal exposures), rabies immunoglobulin (RIG, such as equine RIG or human RIG) should be injected in combination, with infiltration injection around the wound (refer to the "Guidelines for the Prevention and Treatment of Rabies Exposure in China (2023 Edition)").

[0021] This regimen is only effective if initiated within 48 hours of exposure (24 hours for head and face exposure), and the preventive effect decreases significantly after the time window. If clinical symptoms (hydrophobia, pharyngeal muscle spasm, progressive paralysis) have already appeared, the existing PEP regimen is completely ineffective.

[0022] For rabies patients after the onset of symptoms, there are currently no effective treatments worldwide; only supportive therapy (sedation, seizure control, and mechanical ventilation to maintain respiration) can be provided. The only reported treatment, the "Milwaukee regimen" (high-dose ribavirin + acyclovir + phenobarbital + immunoglobulin), has had a success rate of less than 10% in subsequent clinical applications since the first successful case was reported in 2004. Furthermore, its mechanism of action is unclear (antiviral activity has not been confirmed), and it has not been recommended by the WHO (see *The Lancet Infectious Diseases*, 2018, Vol. 18, No. 3, pp. 337-344).

[0023] The core deficiency of existing rabies treatment technologies:

[0024] (1) The post-exposure prophylaxis (PEP) time window is strict: it is only effective within 48 hours after exposure (24 hours for the head and face). People exposed in remote areas or who delay seeking medical treatment often miss the best time for prevention, and their risk of developing the disease increases significantly.

[0025] (2) There is no specific treatment after the onset of the disease: the existing supportive therapy can only relieve symptoms and cannot block viral replication and nerve damage. The mortality rate after the onset of the disease is still close to 100%.

[0026] (3) The Milwaukee regimen has poor practicality: the success rate is extremely low (<10%), the mechanism is unclear, and it involves the combined use of multiple drugs (such as ribavirin, which has the risk of serious hemolytic anemia), so its clinical promotion value is limited;

[0027] (4) Existing drugs cannot penetrate the blood-brain barrier: Rabies virus is a typical neurotropic virus, and most antiviral drugs (such as acyclovir) are difficult to enter the central nervous system and cannot act on rabies virus that has invaded the brain. Summary of the Invention

[0028] Addressing the shortcomings of existing rabies treatments, this invention is the first to discover the anti-rabies virus activity of lapatinib, breaking the limitation of lapatinib's use solely for tumor treatment. Furthermore, through in vitro and in vivo experiments, its role in inhibiting rabies virus replication has been demonstrated for the first time, revealing the potential application value of lapatinib in the early treatment of rabies virus exposure. Therefore, the purpose of this invention is to provide a new use for lapatinib: as a candidate drug for rabies treatment, especially for specific treatment of early rabies virus infection, filling the gap in the lack of specific treatment drugs after rabies virus exposure.

[0029] The technical solution of the present invention is as follows:

[0030] First, this invention provides the use of lapatinib in the preparation of anti-rabies virus drugs.

[0031] Furthermore, the antiviral mechanism of the drug includes inhibiting the intracellular nucleic acid replication of rabies virus and the expression of viral M protein.

[0032] Secondly, the present invention also provides a drug for treating rabies, the drug comprising lapatinib.

[0033] Furthermore, the aforementioned drugs for treating rabies also include pharmaceutically acceptable excipients and carriers.

[0034] Furthermore, the excipients are selected from at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, or colorants.

[0035] Furthermore, the dosage form of the drug is selected from at least one of granules, tablets, pills, capsules, injections, or dispersants.

[0036] This invention clarifies the antiviral effects of drugs at the cellular level as follows:

[0037] The cytotoxicity of lapatinib to N2a cells was determined using the CCK-8 assay. Cytotoxicity assays showed that lapatinib exhibited acceptable toxicity levels to N2a cells within the experimental concentration range (0-10 μM). TCID 50The results showed that lapatinib significantly inhibited RABV replication, with viral titers decreasing as drug concentration increased. Direct immunofluorescence further confirmed that lapatinib significantly reduced the fluorescence signal of the viral M protein, indicating significant inhibition of viral replication. Western blotting results showed that lapatinib dose-dependently reduced viral M protein expression.

[0038] This invention clarifies the antiviral effect of the drug at the in vitro level as follows:

[0039] Treatment efficacy after onset showed that in C57BL / 6J mice, a viral infection model was established by intraperitoneal injection of 80 μL of CVS-11 virus solution. Treatment with lapatinib (60 mg / kg / day, intraperitoneal injection, for 14 consecutive days), starting on day 2 of infection, increased the survival rate of affected mice from 0% (virus control group) to 75%. p <0.001, compared with the virus control group); at the same time, the viral load in the mouse brain (qPCR detection) was reduced by 10^3 times compared with the virus control group, confirming that it can effectively clear the virus in the central nervous system.

[0040] Compared with the prior art, the technical effects of the present invention are as follows:

[0041]

[0042] Regarding the safety of lapatinib for the treatment of rabies: Lapatinib has been used clinically for more than ten years in breast cancer treatment. Its adverse reactions are mainly diarrhea and rash (without serious central nervous system toxicity). Moreover, the dosage used for rabies in this invention (50-100 mg / kg / day in mice; the mouse dose needs to be divided by 12.3 to convert to human dose, and the actual human dose is about 4-8 mg / kg / day) is much lower than the maximum tolerated dose for breast cancer treatment (1250 mg / day, which is about 20 mg / kg / day based on a body weight of 60 kg), thus ensuring greater safety. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0044] Figure 1 The mouse survival rate results provided in Example 1 of the present invention.

[0045] Figure 2 The clinical scoring results of mice provided in Example 1 of the present invention.

[0046] Figure 3The results of mouse weight change provided in Example 1 of this invention.

[0047] Figure 4 The results of qPCR detection of viral load in the mouse brain provided in Example 2 of the present invention.

[0048] Figure 5 The results of Western blotting of RABV-M protein in mouse brain tissue provided in Example 3 of this invention.

[0049] Figure 6 The results of lapatinib's cytotoxicity test against N2a cells provided in Example 4 of this invention are shown.

[0050] Figure 7 The TCID provided in Embodiment 5 of the present invention 50 result.

[0051] Figure 8 The results are Western blotting results provided in Embodiment 5 of the present invention.

[0052] Figure 9 The result is the DFA (direct immunofluorescence) result provided in Example 5 of this invention.

[0053] Figure 10 The effect of lapatinib provided in Example 6 of the present invention on inflammatory factors IL-6, IL-1β, and TNF-α. Detailed Implementation

[0054] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0056] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0057] Example 1: Effect of lapatinib on survival rate of mice with rabies after exposure.

[0058] 1. Experimental Materials

[0059] Drugs: Lapatinib (CAS No.: 231277-92-2) prepared as a solution in PBS (prepare immediately before use); RABV (CVS-11 strain);

[0060] Experimental animals: Female C57BL / 6J mice, 6 - 8 weeks old, weighing 15 - 18 g, with the certificate number SCXK (Beijing) 2024 - 0001, provided by Beijing Ancient Capital Jintai Biotechnology Co., Ltd.;

[0061] Virus: Rabies virus CVS - 11 strain, stored in our laboratory;

[0062] Laboratory conditions: Room temperature 22 - 25 °C, relative humidity 40% - 50%, ventilated by a ventilation fan (ventilated 10 times per hour), natural light source for 12 h per day (8:00 - 20:00), cage - raised (8 mice per cage), the cage was cleaned once every 2 days, the feed was special feed for sterile mice, and free drinking water was provided.

[0063] 2. Experimental procedures

[0064] Grouping: 40 mice were randomly divided into 4 groups, with 10 mice in each group. The specific treatments were as follows:

[0065] Blank control group (PBS): No drug was given and no virus was inoculated, only 80 μL of PBS was injected intraperitoneally;

[0066] Drug - administered alone group (Lapatinib): Only lapatinib was injected intraperitoneally (60 mg / kg, 116 μL / mouse), once a day for 14 consecutive days, and no virus was inoculated;

[0067] Virus - inoculated alone group (RABV+PBS): 0.1 mL of CVS - 11 virus solution was injected intraperitoneally (the day of virus inoculation was recorded as "day 0"), no drug was given, and only 0.1 mL of PBS was injected intraperitoneally;

[0068] Lapatinib - administered group (RABV+Lapatinib): Starting from day 0 after virus inoculation, lapatinib (60 mg / kg) was injected intraperitoneally once a day.

[0069] Observation indicators: Observe and record the status of mice at 9:00 am every day, including:

[0070] Survival status: Record the number of surviving mice in each group every day, and calculate the 14 - day survival rate;

[0071] Clinical symptoms: Record whether the typical symptoms of rabies (ruffled fur, photophobia, increased aggression, hind - limb paralysis, inability to eat and drink independently, difficulty breathing) appeared. When the dying symptoms (difficulty breathing + inability to stand) appeared, the mice were immediately sacrificed (by cervical dislocation).

[0072] Data statistics: Statistical analysis was performed using GraphPad Prism8 software. Survival rate comparisons were performed using a two-tailed unpaired Student's t-test, and multiple group comparisons were conducted using two-way ANOVA combined with Tukey's post-hoc test. Statistical significance was defined as p < 0.05, and the notation was as follows: * p < 0.05, ** p < 0.01.

[0073] 3. Experimental Results and Analysis

[0074] like Figure 1 and Figure 2 As shown, the 14-day survival rate of both the blank control group and the single-drug group was 100%, confirming that lapatinib (60 mg / kg / day) is non-toxic to normal mice; the 14-day survival rate of the single-drug group was 0%, consistent with the pathogenic characteristics of the rabies model; the survival rate of the lapatinib-treated group was 75%, significantly higher than that of group C ( p The result <0.001 indicates that lapatinib has a significant protective effect against rabies virus infection in mice.

[0075] Example 2: Effect of lapatinib on viral load in the brain of mice that developed rabies virus after exposure.

[0076] 1. Experimental Materials

[0077] Same as Example 1, but with the addition of Trizol reagent and RABV-N fluorescent quantitative PCR primers (primers targeting the N gene).

[0078] 2. Experimental Procedure

[0079] Grouping and processing: Same as in Example 1.

[0080] Brain tissue was processed from euthanized mice:

[0081] ① Remove the whole brain of the mouse, rinse with cooled PBS, add 600 μL PBS and grind;

[0082] ② Take 200 μL of the grinding buffer and add 800 μL of Trizol reagent;

[0083] ③Total RNA was extracted using the Trizol RNA extraction reagent method, and the concentration was measured and reverse transcribed.

[0084] ④ qPCR detection of rabies virus RNA in brain tissue.

[0085] 3. Experimental Results and Analysis

[0086] qPCR detection, such as Figure 4 As shown, the N gene of rabies virus was not detected in the blank control group, confirming the absence of viral contamination; the viral load in the group receiving the virus alone was as high as 5.1 log.10 Copies / g of brain tissue indicate that the virus is replicating extensively in the brain; the viral load in the high-dose lapatinib group decreased to 1.5 log. 10 Copies / g of brain tissue (reduced by 3.6 orders of magnitude) confirmed that lapatinib can significantly inhibit viral replication in the brain.

[0087] Example 3: Effect of lapatinib on viral protein expression in mouse brain tissue during early rabies virus exposure

[0088] 1. Experimental Materials

[0089] Same as in Example 1, with the following additional reagents: RIPA (containing protease inhibitor), CBB protein quantification reagent, RABV-M antibody, HPR-labeled goat anti-mouse secondary antibody, and β-actin.

[0090] 2. Experimental Procedure

[0091] Grouping and processing: Same as in Example 2.

[0092] Brain tissue protein extraction and quantification:

[0093] ① Remove the whole brain of the mouse, rinse with cooled PBS, add 600 μL PBS and grind;

[0094] ② Take 200 μL of the grinding buffer, add 400 μL of RIPA reagent, and centrifuge at 12000 rpm for 10 min at 4 ℃;

[0095] ③ Use CBB to quantify brain tissue;

[0096] ④ Western blotting detection.

[0097] 3. Experimental Results and Analysis

[0098] Western blotting results are as follows: Figure 5 As shown, no RABV-M protein was detected in the blank control, confirming the absence of protein contamination; the virus M protein was highly expressed in the single-inoculation group (M protein is a key protein in viral assembly, and its expression level reflects the activity of viral replication); no M protein was detected in the lapatinib administration group, further confirming that lapatinib can inhibit the expression of rabies virus protein, thereby blocking viral replication.

[0099] Example 4: Detection of cytotoxicity of lapatinib

[0100] 1. Experimental Materials

[0101] Lapatinib (CAS No.: 231277-92-2), N2a cells, CCK8 reagent, DMSO, cell culture incubator, etc.

[0102] 2. Experimental Procedure

[0103] ①Prepare cells by plating the cells the day before;

[0104] ② The next day, the drugs (concentrations of 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM) were added and incubated in a 37°C incubator for 24 h.

[0105] ③ After incubation, add 10 μL of CCK8 reagent to each well and incubate at 37℃ for 1~2 h;

[0106] ④Measured values: The measured light wavelength is 480 nm.

[0107] 3. Experimental Results and Analysis

[0108] Cytotoxicity test results as follows Figure 6 As shown, lapatinib exhibits acceptable toxicity levels in N2a cells within the experimental concentration range (0-10 μM).

[0109] Example 5: Effects of lapatinib on viral replication at the cellular level

[0110] 1. Experimental Materials

[0111] Lapatinib (CAS No.: 231277-92-2), HEK-293 cells, RABV-M antibody, etc.

[0112] 2. Experimental Procedure

[0113] ① HEK-293 cells were inoculated with the drug and collected and fixed 48 hours later;

[0114] ② Perform TCID on the collected cell supernatant 50 Measurement:

[0115] ③ Collect cell samples for Western blotting;

[0116] ④ Perform DFA experiments on a fixed sample.

[0117] 3. Experimental Results and Analysis

[0118] TCID 50 The results are as follows Figure 7 As shown, TCID 50 The results showed that lapatinib could significantly inhibit RABV replication, and the viral titer decreased with increasing drug concentration within the experimental concentration range (0-10 μM).

[0119] Western blotting results are as follows: Figure 8 As shown, the results indicate that paltinib can dose-dependently reduce the expression of viral M protein within the experimental concentration range (0-10 μM).

[0120] FDA experimental results as follows Figure 9 As shown, the control group (PBS) results represent the fluorescence signal of cells normally infected with the virus, while the lapatinib group showed a significant reduction in the fluorescence signal of viral proteins after administration, further confirming that lapatinib can significantly reduce the fluorescence signal of viral proteins, indicating that viral replication is significantly inhibited.

[0121] Example 6: Effects of lapatinib on inflammatory factors

[0122] 1. Experimental Materials

[0123] Lapatinib (CAS No.: 231277-92-2), IL-6, IL-1β, TNF-α kits, etc.

[0124] 2. Experimental Procedure:

[0125] ① Prepare the standard curve, prepare the reagent plate, and wash it three times;

[0126] ② Add the sample and incubate at 37 ℃ for 90 min;

[0127] ③ Add 300 μL of washing solution to each well and wash the plate 5 times, patting it dry after each wash;

[0128] ④ Add 100 μL of biotinylate antibody to each well and incubate at 37 °C for 90 min;

[0129] ⑤ Add 300 μL of washing solution to each well and wash the plate 5 times, patting it dry after each wash;

[0130] ⑥ Add 100 μL of enzyme conjugate working solution to each well and incubate at 37 ℃ for 90 min;

[0131] ⑦ Add 300 μL of washing solution to each well and wash the plate 5 times, patting it dry after each wash;

[0132] ⑧ Add 100 μL of colorimetric solution to each well and incubate at 37 °C for 15 min in the dark;

[0133] ⑨ Add stop solution to each well, mix well, and immediately determine the concentration.

[0134] 3. Experimental Results and Analysis

[0135] ELISA results as follows Figure 10 As shown, the expression levels of inflammatory factors in the experimental group (lapatinib + CVS-11) were lower than those in the control group (CVS-11), indicating that lapatinib can reduce the inflammatory response in the brain of virus-infected animals.

[0136] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Application of lapatinib in the preparation of anti-rabies virus drugs.

2. The application as described in claim 1, characterized in that, The antiviral mechanism of the drug includes inhibiting the replication of rabies virus nucleic acid within cells and the expression of the viral M protein.

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