Use of baricitinib in the treatment of chikungunya virus infection

Abstract

The present invention relates to the use of baloxavir sodium and / or solvates thereof and / or hydrates thereof of Formula I, for the preparation of a medicament for the treatment of a Bunyavirus, in particular Crimean-Congo hemorrhagic fever virus (CCHFV) infection,

Description

Technical Field

[0001] This invention relates to the use of baloxavir sodium, a compound of Formula I, and / or its solvates and / or hydrates, in the preparation of medicaments for treating Bunyavirus, particularly Crimean-Congo hemorrhagic fever virus (CCHFV) infection, and also to the use of compositions comprising the compound and / or its solvates and / or hydrates, such as injections, in the preparation of medicaments for treating Bunyavirus, particularly Crimean-Congo hemorrhagic fever virus (CCHFV) infection.

[0002] Background Technology

[0003] Crimean-Congo hemorrhagic fever (CCHF) is an acute infectious disease prevalent in Africa, Asia, and Europe, characterized by fever, gastrointestinal bleeding, hematemesis, and shock, with a mortality rate reaching 50%. The causative agent of CCHF is the Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV belongs to the order Bunyavirales, family Nairoviridae, genus Orthonairovirus, and is a tick-borne virus. CCHF patients exhibit a wide range of clinical symptoms, with a typical course consisting of four stages: incubation period, pre-hemorrhagic phase, hemorrhagic phase, and recovery phase. The length of the incubation period is related to factors such as the route of exposure and viral load. After the incubation period, the pre-hemorrhagic phase manifests as fever, accompanied by severe headache, nausea, diarrhea, muscle aches, and photophobia. As the patient progresses to the hemorrhagic phase, petechiae, large ecchymoses, and massive hemorrhage appear sequentially, with 9%–50% of cases resulting in death during this stage. In addition to these symptoms, studies have also found that CCHF is accompanied by severe damage to the liver, nervous system, respiratory system, and heart. Currently, there are no marketed drugs or vaccines for Crimean-Congo hemorrhagic fever. Summary of the Invention

[0004] The purpose of this invention is to discover drugs with antiviral activity against Bunyaviruses, particularly Crimean-Congo hemorrhagic fever virus, which can be used to treat related diseases caused by this virus, such as fever, gastrointestinal bleeding, hematemesis, and shock. Baloxaviric acid exhibits anti-Criminal-Congo hemorrhagic fever virus activity in vitro, but its solubility is low, making it unsuitable for injection. This invention, through inventive research, has discovered that the compound shown in Formula I, baloxavir sodium, has good solubility and exhibits inhibitory activity against Crimean-Congo hemorrhagic fever virus both in vivo and in vitro. It can be used to prepare injectable formulations and shows good therapeutic effects in treating diseases caused by Crimean-Congo hemorrhagic fever virus.

[0005] This invention provides compounds having the structure of Formula I and / or their solvates and / or their hydrates:

[0006]

[0007] The compound shown in Formula I can inhibit viral replication on cells and reduce viral nucleic acid load in cell cultures.

[0008] After creative invention and research, the inventors of this invention discovered some new functional characteristics of the compound of formula I:

[0009] First, the compound shown in Formula I can reduce the viral nucleic acid load level in cells infected with Crimean-Congo hemorrhagic fever at micromolar concentrations.

[0010] Second, the compound shown in Formula I has a significant protective effect against Crimean-Congo hemorrhagic fever virus (CCHFV) infection in mice.

[0011] This invention relates to the use of compounds of Formula I and / or their solvates and / or hydrates in the preparation of pharmaceuticals for treating diseases or infections caused by Bunyavirus in subjects.

[0012]

[0013] The present invention also relates to the use of the compound of Formula I and / or its solvates and / or its hydrates in the preparation of a medicament as a Bunyavirus inhibitor.

[0014] The present invention also relates to the use of the compounds of Formula I and / or their solvates and / or their hydrates in the preparation of medicaments for inhibiting the replication or reproduction of Bunyavirus in cells (e.g., mammalian cells) in vivo or in vitro.

[0015] The present invention also relates to the use of the composition in the preparation of a medicament for treating a disease or infection caused by a Bunyavirus in a subject.

[0016] The present invention also relates to the use of the composition in the preparation of a medicament as a Bunyavirus inhibitor.

[0017] The present invention also relates to the use of the composition in the preparation of a medicament for inhibiting the replication or reproduction of Bunyavirus in cells (e.g., mammalian cells) in vivo or in vitro.

[0018] In some embodiments, the composition comprises a compound of Formula I and / or its solvate and / or its hydrate. In some embodiments, the compound of Formula I and / or its solvate and / or its hydrate is the active ingredient. In some embodiments, the compound of Formula I and / or its solvate and / or its hydrate is present in an effective amount. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition is an injectable preparation.

[0019] In some embodiments, the injectable comprises:

[0020] (1) The content of compound I and / or its solvates and / or hydrates shown in formula (1) is 0.1-20%;

[0021] (2) Solvent, selected from water for injection, physiological saline, sterile water, etc., with a content of 60-99%;

[0022] (3) Selected from one or more of the following excipients:

[0023] pH adjusters, such as hydrochloric acid and acetic acid, are present in concentrations of 0.01-5%.

[0024] Isotonic agents, such as mannose and sodium chloride, are present in a concentration of 0.1-10%.

[0025] Preservatives, such as parabens, are present in a concentration of 0.01-1%.

[0026] Stabilizers, such as ethanol and propylene glycol, are present in a concentration of 0.1-10%.

[0027] In some embodiments, the pH of the injectable is 4 to 9.

[0028] The present invention also relates to the use of the injection in the preparation of a medicament for treating a disease or infection caused by a Bunyavirus in a subject.

[0029] The present invention also relates to the use of the injection in the preparation of a medicament for inhibiting the replication or reproduction of Bunyavirus in cells (e.g., mammalian cells) in vivo or in vitro.

[0030] The present invention also relates to the use of the injection in the preparation of a medicament as a Bunyavirus inhibitor.

[0031] In some embodiments, the compound represented by Formula I and / or its solvates and / or its hydrates are administered to the subject as the active ingredient via injection.

[0032] In some embodiments, the compound of Formula I and / or its solvates and / or hydrates, or the composition or the drug, is preferably administered to the subject by injection, such as intravenous, intramuscular or other injection methods.

[0033] In some embodiments, the compound of Formula I and / or its solvates and / or its hydrates, or the composition thereof, or the drug thereof, is administered to the subject by injection at a dose of 1 mg / kg to 1000 mg / kg body weight (based on the active ingredient).

[0034] In some implementations, the Bunyavirus is a virus from the Naroviridae family.

[0035] In some implementations, the Bunyavirus is a virus belonging to the genus Nerovirus.

[0036] In some implementations, the Bunyavirus is Crimean-Congo hemorrhagic fever virus (CCHFV).

[0037] In some embodiments, the illness caused by the Bunyavirus is a simple infection caused by the Bunyavirus, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, nosebleeds, hematemesis, or shock.

[0038] In some implementations, the disease caused by the Bunyavirus is hemorrhagic fever caused by a Naroviridae virus.

[0039] In some implementations, the disease caused by the Bunyavirus is hemorrhagic fever caused by a virus of the genus Nerovirus.

[0040] In some implementations, the disease caused by the Bunyavirus is hemorrhagic fever caused by Crimean-Congo hemorrhagic fever virus.

[0041] In some implementations, the disease caused by the Bunyavirus is Crimean-Congo hemorrhagic fever.

[0042] In some embodiments, the mammals include bovids, equines, caprines, suidae, canids, felines, rodents, primates, such as humans, cats, dogs, or pigs.

[0043] In some implementations, the subjects include bovines, equines, caprines, suidae, canines, felines, rodents, primates, such as humans, cats, dogs, or pigs.

[0044] The present invention also relates to a method for treating and / or preventing disease or infection in a subject in need, or for inhibiting the replication or reproduction of Bunyavirus in a subject in need, the method comprising administering to the subject in need a therapeutic and / or preventive effective amount of the composition comprising the compound of Formula I and / or its solvates and / or its hydrates, or the compound of Formula I and / or its solvates and / or its hydrates, wherein the disease includes a disease or infection caused by Bunyavirus.

[0045] In this invention, the terms "therapeutic effective amount" or "preventive effective amount" refer to an amount sufficient, within reasonable medical judgment, to treat or prevent a patient's disease while avoiding serious side effects with a sufficiently low risk-reward ratio. The therapeutic effective amount of a compound will vary depending on factors such as the specific compound chosen (e.g., considering the compound's potency, effectiveness, and half-life), the chosen route of administration, the disease being treated, the severity of the disease, the patient's age, size, weight, and physical condition, the patient's medical history, the duration of treatment, the nature of concurrent therapies, and the desired therapeutic effect, but can still be conventionally determined by those skilled in the art.

[0046] It should also be noted that the specific dosage and method of administration of the compound represented by Formula I and / or its solvates and / or hydrates for different patients depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, drug activity, timing of administration, metabolic rate, severity of illness, and the subjective judgment of the treating physician. A dosage between 0.001 and 1000 mg / kg body weight / day is preferred, for example, a dosage from 1 mg / kg to 1000 mg / kg body weight / day.

[0047] The compositions of the present invention can be prepared in various forms according to different routes of administration, such as injections, including powder injections and solutions. Attached Figure Description

[0048] Figure 1 The results of the in vitro anti-Crimea-Congo hemorrhagic fever virus experiment using baloxavir sodium of the present invention;

[0049] Figure 2 The results of the in vivo pharmacodynamic experiments of baloxavir sodium in mice are as follows. Detailed Implementation

[0050] The following embodiments are illustrative preferred embodiments of the present invention and do not constitute any limitation on the present invention.

[0051] Example 1: Preparation of Baloxavir Sodium Salt

[0052] 4.9 g (0.01 mol) of baloxaviric acid was added to 20 mL of water, followed by 20 mL (0.02 mol) of 1 N NaOH. The reaction was carried out at 80 °C for 12 h. The reaction was then stopped, and the product was filtered to obtain 4.5 g of a white solid, with a yield of 88.8%.

[0053] Example 2: Study on the solubility of baloxavir sodium

[0054] According to the Chinese Pharmacopoeia (2020 Edition), the equilibrium solubility in pure water was determined using the room temperature shake-flask method. The sodium baloxavir used in this study had a purity of 99.5%. The powdered sample was weighed and placed in a specific volume of solvent, and shaken thoroughly for 30 seconds every 5 minutes at room temperature. The dissolution behavior of the sample was observed after 30 minutes. If no solute particles were observed, the solute was considered completely dissolved. A control example was also established to compare the solubility of sodium baloxavir, mabaloxavir, and baloxavir.

[0055]

[0056] As shown in Table 1, baloxavir sodium has high water solubility.

[0057] Table 1. Compound solubility test

[0058]

[0059] Example 3: In vitro screening experiment of baloxavir sodium against Crimean-Congo hemorrhagic fever virus

[0060] 3.1 Drug treatment of virus-infected cells

[0061] Human umbilical vein endothelial cells (HUVECs) were cultured in Eagle medium (Gibco, Rockville, MD, USA) at 37°C with a minimum CO2 concentration of 5% and supplemented with 10% fetal bovine serum (Gibco). The CCHFV strain IbAr10200-eGFP used in the study was stored at the National Virus Resource Center (Wuhan, China). All CCHFV infection experiments were performed in a BLS-3 laboratory.

[0062] 3.2 Cytotoxicity assay

[0063] Healthy HUVEC cells were divided into approximately 1 × 10⁻⁶ cells. 4Seeds were seeded per well in 96-well plates and incubated overnight at 37°C. The next day, the plates were ready for use. Different drugs were diluted with DMEM medium containing 2% FBS in 3-fold or 2-fold serial dilutions, 1 ml per concentration, for a total of 7 serial dilutions. Each drug concentration was applied in triplicate. The remaining drug-containing medium was stored at 4°C. 10 μL of CCK-8 solution (Beyotime, China) was added to each well. The plates were incubated for 1-4 hours, and the absorbance at 450 nm was measured using a microplate reader to determine the median cytotoxic concentration (Cc). 50 ).

[0064] 3.3 Viral load detection

[0065] To evaluate the activity of antiviral drugs, HUVEC cells (approximately 6 × 10⁶ cells) were used. 4 Cells were pre-seeded in 48-well plates with serially diluted drug, and incubated at 37°C for 1 hour for infection. After infection at 37°C with a multiple infection index (MOI) of 0.01 for 1 hour, the cell supernatant was discarded, and cells were cultured in fresh drug-containing medium. At 72 hours post-infection (pi), 100 μL of supernatant was collected for RNA extraction, and cells were subjected to IFA or Western blot analysis. The RNA copy number in the supernatant was then quantified using real-time quantitative polymerase chain reaction (qRT-PCR), and the median effective concentration (EC50) was calculated. 50 The primers for amplifying the CCHFVS segment are: (qF)5′-TCAAGTGGAGGAAGGACATAGG-3′ and (qR)5′-TCCACATGTTCACGGCTCACTGGG-3′.

[0066] Total RNA was extracted using TRIzol reagent (Takara) according to the manufacturer's instructions. qRT-PCR was performed using 1 μg of total RNA as described above.

[0067] 3.4 Experimental Results

[0068] To preliminarily evaluate the in vitro anti-CCHFV effect of baloxavir sodium, a screening test was conducted using two drug concentrations (20 μM and 5 μM) of baloxavir sodium and the positive control T-705. HUVEC cells were infected in the presence of either drug or DMSO, and fluorescence images were captured every 48 hours to quantify the infection rate. At concentrations of 20 μM and 5 μM, baloxavir sodium showed inhibition rates of approximately 100% and 90% against CCHFV infection, respectively. At the same concentrations, T-705 only inhibited approximately 80% and 30% of viral infection, respectively.

[0069] Furthermore, the EC50 of baloxavir sodium and T-705 was quantitatively determined by qRT-PCR. 50 .like Figure 1 As shown, Baloxavir's EC 50 The value was low, at 0.6 μM, while the EC value of T-705 was... 50 The value was 4.2 μM. The 50% cytotoxic concentration (CFC) of baloxavir sodium and T-705 was... 50 The selectivity indices were 83.6 μM and >100 μM, respectively, with selectivity indices (SI = CC). 50 / EC 50 The values ​​were 139.3 and >23.8, respectively. These results indicate that, under these infection conditions, baloxavir sodium exhibits better anti-CCHFV activity in vitro compared to T-705.

[0070] Example 4: Pharmacokinetic Study of Baloxavir Sodium in Rats

[0071] Male SD rats were used in the experiment and divided into an oral administration group and an intraperitoneal injection group. Baloxavir sodium and Tween 80 were prepared into injection solutions and administered accordingly. Blood samples were collected at different time points after administration (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 12.083 h, 12.25 h, 12.5 h, 13 h, 14 h, and 24 h). Whole blood was centrifuged at 2500 G for 10 min at 4 °C within 2 h, separated and placed into EP tubes, and stored in a -20 °C refrigerator. For sample analysis, 10 μl of plasma was added to 10 μl of blank plasma and vortexed. Then, 20 μl of acetonitrile and 100 μl of acetonitrile precipitant (containing 5 ng / ml propranolol acetonitrile solution) were added. The sample was shaken for 1 min and centrifuged at 14000 rpm for 10 min at 4℃. 100 μl of the supernatant was centrifuged and 400 μl of diluent (50% acetonitrile aqueous solution) was added. After thorough shaking, the sample was analyzed using LC-MS / MS8050.

[0072] In SD rats, the pharmacokinetic characteristics of baloxavir sodium were observed after administration. Table 2 shows the pharmacokinetic characteristics compared to orally administered baloxavir sodium (AUC). 0-t=0.544±0.269μg·h / mL; C max Compared to (0.062 ± 0.022 μg / mL), the plasma exposure of baloxavir sodium after intraperitoneal injection at a dose of 75 mg / kg was AUC. 0-t =23.49±6.08 μg·h / mL and maximum plasma concentration C max =1.82±0.5μg / mL. These results indicate that intraperitoneal injection of baloxavir sodium has favorable pharmacokinetic properties in rats.

[0073] Table 2 Pharmacokinetic Tests of Compounds

[0074]

[0075] Example 5: Experiment on the protection of mice infected with Crimean-Congo hemorrhagic fever virus (CCHFV) from death by baloxavir sodium injection.

[0076] All animal experiments used type I IFN receptor knockout (IFNAR) on a C57BL / 6J background. - / - Mice. Female mice aged 7-12 weeks were infected with CCHFV-YL16070 strain (3000 TCID) via intraperitoneal injection (IP). 50 Mice were administered baloxavir sodium via intraperitoneal injection (IP), while the control group received a similar solvent injection. Mouse weight and clinical signs were monitored daily. Mice were euthanized with isoflurane anesthesia when they lost more than 20% of their body weight, became unresponsive to touch, and had difficulty crawling. Statistical analysis was performed using two-way ANOVA. Mouse livers were collected for viral load determination and pathological analysis. Survival percentages (P-values) were determined using a log-rank test. Statistical analysis was performed using unpaired t-tests.

[0077] We evaluated the in vivo efficacy of baloxavir sodium against CCHFV. Baloxavir sodium Tween 80 injection was administered intraperitoneally twice daily at a dose of 75 mg / kg for 3 days. The treatment group showed a protective effect against CCHFV infection in mice. The study showed that the overall survival rate of the 75 mg / kg twice daily regimen was 83.3%. Figure 2 The survival rate of the control group (n=6 mice) was 16.7%, while that of the control group was 16.7%. After treatment, the viral load in the liver of mice administered 75 mg / kg twice daily decreased by approximately 2 log copies / mg tissue on day 3 (*P<0.05) and day 6 (*P<0.01). Figure 2 (C, D). Compared with the control group, the weight loss in mice treated with baloxavir sodium was also alleviated. Figure 2 (B).

Claims

1. Use of the compound represented by Formula I in the preparation of a medicament for treating a disease or infection caused by a Bunyavirus in a subject. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

2. Use of the composition in the preparation of a medicament for treating a disease or infection caused by a Bunyavirus in a subject, wherein the composition comprises a compound of Formula I. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

3. Use of the composition in the preparation of a medicament as a Bunyavirus inhibitor, wherein the composition comprises a compound represented by Formula I. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

4. Use of the composition in the preparation of a medicament for inhibiting the replication or reproduction of Bunyavirus in cells, either in vivo or in vitro, wherein the composition comprises a compound represented by Formula I. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

5. The use according to any one of claims 2-4, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

6. The use according to claim 5, wherein the composition is an injectable preparation.

7. The use according to claim 6, wherein the injectable comprises: (1) The content of compound I shown in formula is 0.1-20%; (2) Solvent, selected from water for injection, physiological saline, or sterile water, with a content of 60-99%; (3) Selected from one or more of the following excipients: pH adjuster, with a content of 0.01-5%; Isotonic agents, with a content of 0.1-10%; Preservatives, with a content of 0.01-1%; Stabilizer, with a content of 0.1-10%.

8. The use according to claim 7, wherein the pH adjuster is selected from hydrochloric acid and acetic acid; the isotonic agent is selected from mannose and sodium chloride; the preservative is paraben; and the stabilizer is selected from ethanol and propylene glycol.

9. The use according to any one of claims 6-8, wherein the pH value of the injection is 4-9.

10. Use of the compound represented by Formula I in the preparation of a drug as a Bunyavirus inhibitor. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

11. Use of the compound represented by Formula I in the preparation of a medicament for inhibiting the replication or reproduction of Bunyavirus in cells, either in vivo or in vitro. Formula I The Bunyavirus mentioned above is the Crimean-Congo hemorrhagic fever virus.

12. The use according to any one of claims 1-4, 10-11, wherein the drug is administered to the subject by injection.

13. The use of claim 12, wherein the drug is administered to the subject intravenously or intramuscularly.

14. The use according to claim 12, wherein the drug is administered to the subject by injection at a dose of 1 mg active ingredient / kg to 1000 mg active ingredient / kg body weight.

15. The use according to claim 1 or 2, wherein the disease caused by the Bunyavirus is a simple infection caused by Bunyavirus, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis, hematemesis, or shock; or The disease caused by the Bunyavirus is hemorrhagic fever caused by the Crimean-Congo hemorrhagic fever virus.

16. The use as claimed in claim 1 or 2, wherein the disease caused by the Bunyavirus is Crimean-Congo hemorrhagic fever.

17. The use as claimed in claim 4 or 11, wherein the cell is a mammalian cell.

18. The use according to claim 17, wherein the mammal is a bovine, equine, suidae, canine, feline, rodent, or primate.

19. The use of claim 18, wherein the mammal is a human, cat, dog, or pig.

20. The use as claimed in claim 18, wherein the mammal is a caprine animal.

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