CN108853106B - Use of imine phenazine compound as rabies virus inhibitor - Google Patents
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Abstract
The invention belongs to the technical field of medicines. In particular, the invention relates to a composition of formula (I)(I) Use of structural iminophenazine compounds for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection (e.g. rabies). The application also relates to the use of the iminophenazine compounds for preparing a pharmaceutical composition for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection (e.g. rabies).
Description
Technical Field
The invention belongs to the technical field of medicines. In particular, the invention relates to the use of the iminophenazine compounds having the structure of formula (I) for the prevention and/or treatment of rabies virus infection or diseases caused by rabies virus infection (e.g. rabies). The application also relates to the use of the iminophenazine compounds for preparing a pharmaceutical composition for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection (e.g. rabies).
Background
Rabies is an infectious disease of both human and veterinary co-morbidities caused by rabies virus, and is susceptible to almost all warm-blooded animals, of which canines are the main hosts and play an important role in the transmission of disease. The main transmission route is through animal bite, virus in saliva enters into body through damaged skin, and other routes are through digestive tract and animal scratch.
In recent years, the reported death number of rabies reports that infectious diseases are the prostate in China, and brings serious threat to the life health of people. Around 2,000 people are reported to be infected with rabies annually, however, due to the lack of effective drugs and therapeutic measures, the mortality rate of rabies patients is almost 100%.
At present, two biological products for preventing and treating rabies mainly comprise vaccine and immunoglobulin, and are difficult to widely use due to higher price and low-temperature storage. Especially in rural areas in China, many families have housedogs, but few dogs raised artificially are immunized, self-protection consciousness is thin, and dogs are too close and have lucky psychology, so that the pet can not suffer from rabies, and after some people are bitten by dogs and cats, the immune globulin and the vaccine are high in price and do not receive vaccination, and finally, the disease is caused to die. The chemical medicine can be placed at normal temperature, is convenient to store and low in cost, can be widely used in a large scale in China, and the prevention and treatment of rabies is not brought into the range of major animal epidemic diseases in China, so that the search for common clinical medicines for resisting rabies virus is urgent.
Disclosure of Invention
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of cell culture, biochemistry, cell biology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
As used herein, the term "Rabies Virus (RV)" refers to a negative strand RNA virus of the Rhabdoviridae (Rhabdoviridae) Lyssavirus (Lyssavirus) genus, which is the causative agent of Rabies.
As used herein, the term "pharmaceutically acceptable salt" refers to salts of basic functional groups (e.g., -NH-) present in the compounds provided herein with suitable inorganic or organic anions (acids), and includes, but is not limited to, hydrohalic salts, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, and the like; inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate and the like; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate and the like; aryl sulfonates such as benzenesulfonate, p-benzenesulfonate and the like; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.; amino acid salts such as glycinate, trimethylglycinate, arginate, ornithine, glutamate, aspartate and the like.
As used herein, the expression "Cx-CyAlkyl "refers to a saturated aliphatic group having a specific range of carbon atoms. For example, the expression "C1-C4Straight chain alkyl "refers to straight chain alkyl groups comprising 1 to 4 carbon atoms, specific examples include, but are not limited to, methyl, ethyl, n-propyl, or n-butyl; for example, the expression "C1-C4Branched alkyl "refers to a branched alkyl group comprising 1 to 4 carbon atoms, specific examples include, but are not limited to, isopropyl, tert-butyl, or isobutyl.
As used herein, the term "halo Cx-CyAlkyl "means that one or more (e.g. 2, 3 or 4) halogen atoms are substituted for Cx-CyGroups derived from one or more (e.g. 2, 3 or 4) hydrogen atoms on the alkyl group.
An "effective amount" as used herein means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, a "disease-preventing effective amount" refers to an amount sufficient to prevent, or delay the onset of disease; by "a disease-treating effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
The inventor of the application discovers for the first time that the imine phenazine compound with a specific structure can neutralize rabies virus, remarkably inhibit the infection of the rabies virus, has obvious anti-rabies virus activity, and can be used for preventing and/or treating rabies virus infection or diseases (such as rabies) caused by the rabies virus infection. Before the present application, the anti-rabies virus activity of the compounds has not been reported.
Accordingly, in one aspect, the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection in a subject, or for the preparation of a pharmaceutical composition for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection in a subject;
wherein the content of the first and second substances,
R1、R3each independently is halogen (e.g., -F, -Cl, -Br, or-I);
R2is C1-C4Alkyl, or halo C1-C4An alkyl group.
In certain preferred embodiments, R1、R3Each independently is-F, -Cl, -Br or-I.
In certain preferred embodiments, R1And R3The same is true.
In certain preferred embodiments, R1And R3is-Cl.
In certain preferred embodiments, R2Is C1-C4Straight chain alkyl or C1-C4A branched alkyl group; optionally, wherein said C1-C4Straight chain alkyl or C1-C4Each branched alkyl group is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: halogen (e.g., -F, -Cl, -Br, or-I).
In certain preferred embodiments, R2Is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; optionally wherein each of said groups is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: -F, -Cl, -Br or-I.
In certain preferred embodiments, R2Is C1-C3Straight chain alkyl or C1-C3A branched alkyl group; optionally, wherein said C1-C3Straight chain alkyl or C1-C3Each branched alkyl group is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: halogen (e.g., -F, -Cl, -Br, or-I).
In certain preferred embodiments, R2Is methyl, ethyl, n-propyl or isopropyl; optionally wherein each of said groups is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: -F, -Cl, -Br or-I.
In certain preferred embodiments, R2Is isopropyl.
In certain preferred embodiments, the compound is 10- (p-chlorophenyl) -2, 10-dihydro-3- (p-chloroanilino) -2-isopropyliminophenazine.
In certain preferred embodiments, the compounds have the formula shown below:
in certain preferred embodiments, the disease caused by rabies virus infection is rabies.
In certain preferred embodiments, the rabies virus is serotype 1.
In certain preferred embodiments, the pharmaceutical composition comprises a prophylactically or therapeutically effective amount of a compound as described above. In certain preferred embodiments, the pharmaceutical composition may be in any form known in the medical arts. For example, the pharmaceutical composition may be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, and the like.
In certain preferred embodiments, the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent having anti-rabies virus and/or anti-rabies activity. In certain preferred embodiments, the additional pharmaceutically active agent is selected from rabies vaccine, anti-rabies serum or anti-rabies immunoglobulin (e.g., anti-rabies human immunoglobulin).
In certain preferred embodiments, the pharmaceutical composition comprises a unit dose of a compound as described above, for example comprising any amount of said compound from 1 to 2000mg, for example from 10 to 1500mg, for example from 20 to 1000mg, for example from 50 to 500mg, for example from 50 to 100 mg. In the present invention, the dosage of the compound to be administered may be adjusted depending on factors such as the severity of the condition of the patient or subject, age, weight, sex, administration mode and treatment course.
In the present invention, the pharmaceutical composition may be administered by any suitable method known in the art, such as oral, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition may be formulated into oral preparations, for example, oral solid preparations such as tablets, capsules, pills, granules and the like; or oral liquid preparations such as oral solution, oral suspension, syrup, etc. When formulated into oral preparations, the pharmaceutical composition may further comprise suitable fillers, binders, disintegrants, lubricants, etc.; in particular, in order to improve the wettability and to increase the solubility of the hydrophobic compound, the pharmaceutical composition preferably comprises a hydrophilic polymer, such as hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC), or the like. When used parenterally, the pharmaceutical composition may be prepared into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When prepared into injections, the pharmaceutical composition may be manufactured by a conventional method in the existing pharmaceutical field. When an injection is prepared, the pharmaceutical composition may contain a pharmaceutically acceptable carrier such as sterile water, ringer's solution and isotonic sodium chloride solution, and may be supplemented with appropriate additives such as antioxidants, buffers and bacteriostats, depending on the nature of the drug. When used for rectal administration, the pharmaceutical composition may be formulated as suppositories and the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalant or a spray.
In certain preferred embodiments, the subject is a mammal, e.g., a human.
In another aspect, the present invention provides a method for preventing and/or treating rabies virus infection or a disease caused by rabies virus infection, comprising administering to a subject in need thereof an effective amount of a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof;
wherein the content of the first and second substances,
R1、R3each independently is halogen (e.g., -F, -Cl, -Br, or-I);
R2is C1-C4Alkyl, or halo C1-C4An alkyl group.
In certain preferred embodiments, R1、R3Each independently is-F, -Cl, -Br or-I.
In certain preferred embodimentsIn the embodiment, R1And R3The same is true.
In certain preferred embodiments, R1And R3is-Cl.
In certain preferred embodiments, R2Is C1-C4Straight chain alkyl or C1-C4A branched alkyl group; optionally, wherein said C1-C4Straight chain alkyl or C1-C4Each branched alkyl group is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: halogen (e.g., -F, -Cl, -Br, or-I).
In certain preferred embodiments, R2Is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; optionally wherein each of said groups is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: -F, -Cl, -Br or-I.
In certain preferred embodiments, R2Is C1-C3Straight chain alkyl or C1-C3A branched alkyl group; optionally, wherein said C1-C3Straight chain alkyl or C1-C3Each branched alkyl group is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: halogen (e.g., -F, -Cl, -Br, or-I).
In certain preferred embodiments, R2Is methyl, ethyl, n-propyl or isopropyl; optionally wherein each of said groups is independently substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from: -F, -Cl, -Br or-I.
In certain preferred embodiments, the compound is 10- (p-chlorophenyl) -2, 10-dihydro-3- (p-chloroanilino) -2-isopropyliminophenazine.
In certain preferred embodiments, the compounds have the formula shown below:
in certain preferred embodiments, the disease caused by rabies virus infection is rabies.
In certain preferred embodiments, the rabies virus is serotype 1.
In certain preferred embodiments, the compounds described above may be formulated and administered as pharmaceutical compositions. In certain preferred embodiments, the pharmaceutical composition may comprise a prophylactically or therapeutically effective amount of a compound as described above. In certain preferred embodiments, the pharmaceutical composition may be in any form known in the medical arts. For example, the pharmaceutical composition may be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, and the like.
In the present invention, the pharmaceutical composition may be administered by any suitable method known in the art, such as oral, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition may be formulated into oral preparations, for example, oral solid preparations such as tablets, capsules, pills, granules and the like; or oral liquid preparations such as oral solution, oral suspension, syrup, etc. When formulated into oral preparations, the pharmaceutical composition may further comprise suitable fillers, binders, disintegrants, lubricants, etc.; in particular, in order to improve the wettability and to increase the solubility of the hydrophobic compound, the pharmaceutical composition preferably comprises a hydrophilic polymer, such as hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC), or the like. When used parenterally, the pharmaceutical composition may be prepared into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When prepared into injections, the pharmaceutical composition may be manufactured by a conventional method in the existing pharmaceutical field. When an injection is prepared, the pharmaceutical composition may contain a pharmaceutically acceptable carrier such as sterile water, ringer's solution and isotonic sodium chloride solution, and may be supplemented with appropriate additives such as antioxidants, buffers and bacteriostats, depending on the nature of the drug. When used for rectal administration, the pharmaceutical composition may be formulated as suppositories and the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalant or a spray.
In certain preferred embodiments, the method optionally further comprises administering an additional pharmaceutically active agent having anti-rabies virus and/or anti-rabies activity. Such additional pharmaceutically active agents may be administered prior to, concurrently with, or subsequent to the administration of the compound as described above.
In certain preferred embodiments, the additional pharmaceutically active agent is selected from rabies vaccine, anti-rabies serum or anti-rabies immunoglobulin (e.g., anti-rabies human immunoglobulin).
In certain preferred embodiments, a compound as described above may be administered in any amount of 1-2000 mg/kg body weight of the subject, for example, 1-1500 mg/kg body weight/day, 1-1000 mg/kg body weight/day, 5-500 mg/kg body weight/day, 5-200 mg/kg body weight/day, 5-100 mg/kg body weight/day, or 10-100 mg/kg body weight/day. In certain preferred embodiments, a compound as described above may be administered 4 times daily, 3 times daily, 2 times daily, 1 time every two days, or 1 time weekly, optionally repeating a dosing regimen as described above weekly or monthly as appropriate. In the present invention, the dosage of the compound to be administered may be adjusted depending on factors such as the severity of the condition of the patient or subject, age, weight, sex, administration mode and treatment course.
In certain preferred embodiments, the subject is a mammal, e.g., a human.
Advantageous effects of the invention
At present, due to the lack of effective medicines and treatment means for rabies virus infection or rabies, the fatality rate of rabies patients is close to 100%. The inventor of the application discovers the compound with the anti-rabies virus activity for the first time through a great deal of research and screening. The compound can neutralize rabies virus, thereby obviously interfering the infection process of the rabies virus to cells, having obvious protective effect on animal models infected by the rabies virus and having obvious anti-rabies virus activity. Therefore, the compounds can be used for preventing and/or treating rabies virus infection or diseases (such as rabies) caused by the rabies virus infection, and the discovery has great significance for preventing and treating the rabies virus infection and/or rabies.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1 shows the analysis curve of the inhibition rate of RV-07 on rabies pseudovirus in example 2. Wherein the abscissa represents the action concentration (. mu.M) of RV-07 and the ordinate represents the inhibition ratio (%). The results show that RV-07 can obviously inhibit rabies pseudovirus.
FIG. 2 shows the cytotoxicity assay curve of RV-07 in example 3. Wherein the abscissa represents the action concentration (. mu.M) of RV-07 and the ordinate represents the inhibition ratio (%). The results show that RV-07 has no obvious cytotoxicity.
FIG. 3 shows the results of analysis of neutralizing activity of RV-07 against rabies virus in example 4 by fluorescence microscopy. The results show that RV-07 has obvious rabies virus neutralization activity.
FIG. 4 shows the analysis curve of the inhibition rate of RV-07 on rabies virus in example 4. Wherein the abscissa represents the action concentration (. mu.M) of RV-07 and the ordinate represents the inhibition ratio (%). The results show that RV-07 can obviously inhibit the infection of the cells by the rabies live virus.
FIG. 5 shows the results of an analysis of the antiviral activity of RV-07 in the in vivo model of rabies virus infection in example 5. Wherein, A-D show the survival curves of rabies virus infected golden yellow mice treated with different doses (50. mu.l or 15. mu.l) of RV-07 orally (oral) or Intramuscularly (IM), respectively. The result shows that RV-07 can obviously delay the morbidity and the death time of golden hamster and improve the survival rate of golden hamster infected with rabies virus.
Detailed Description
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
In the following examples of the invention, RV-07 refers to clofazimine (10- (p-chlorophenyl) -2, 10-dihydro-3- (p-chlorophenylamino) -2-isopropyliminophenazine) having the formula shown below:
example 1 construction of pseudoviruses
1.1 pseudovirus-based antiviral drug screening principle
For enveloped viruses (e.g., rabies virus), the process of invasion into cells is determined by their envelope proteins, since the envelope is responsible for recognizing receptors on the surface of the target cell and initiating the processes of adsorption and penetration. Furthermore, the phenotypical mixing that occurs when two viruses infect a cell simultaneously suggests that the envelope of one virus can integrate into the surface of the particle of another, different virus. Based on the above two points, a new technology, namely a pseudovirus technology, is generated in the aspects of researching the process of virus invading cells, tissue tropism and receptors thereof, and the like.
A pseudovirus is a virus in which a retrovirus integrates envelope glycoproteins of another different virus species to form an envelope with an exogenous virus, and the genome retains the genomic properties of the retrovirus itself. The pseudovirion is a virus vector recombinant which is provided by a packaging cell and carries a virus protein wrapped with an exogenous gene, has the capability of infecting target cells once, is replication-defective, and cannot generate new virion after infection. Although this virus can invade cells, it is very safe because it is replication-defective after invading cells, and structural proteins assembled into the virus are supplied from an external source, and thus self-replication of the virus cannot be performed. The pseudovirus has the same autophagy and infection process as the euvirus, so the early process of virus infection can be simulated, and the pseudovirus carries a reporter gene (luciferase), so that various detections and analyses can be rapidly and conveniently carried out, such as research on the relationship between the virus and host cells, cloning of virus receptors, screening of antiviral drugs, evaluation of vaccine immunity effect and the like.
The plasmid for expressing rabies virus glycoprotein and the skeleton plasmid are used for cotransfecting 293T cells, so that the rabies pseudovirus is obtained. After the pseudovirus and the drug diluted by times act for a period of time, pseudovirus sensitive cells are added, and 5% CO is added at 37 DEG C2After further culturing in the incubator for 48 hours, the luminescence value was measured. If the drug prevents the virus from adsorbing and entering the cell, the luminescence value of the sample hole can be attenuated or disappeared; if the drug has no anti-viral activity, the luminescence value of the sample well is equivalent to the luminescence value of the virus control.
1.2 preparation of pseudoviruses
a) Preparation of rabies pseudovirus
The envelope plasmid pCAG.CVS and the backbone plasmid pSG 3. delta. env. cmvFluc (see Nie J, et al. Sci Rep.2017 Feb 20; 7:42769.) were co-transfected into 293T cells (ATCC No. CRL3216) at a mass ratio of 3:1 using the transfection reagent Lipofectamine 2000, the medium was changed about 4-6h after transfection, and then placed at 37 deg.C，5％CO2Culturing in incubator for about 48 h. The transfected cell culture medium supernatant was collected, centrifuged, cell debris removed, filtered through a 0.45 μm filter, and dispensed into 1.5mL centrifuge tubes and stored in a-80 ℃ freezer for use.
b) Preparation of Vesicular Stomatitis Virus (VSV) pseudovirus
The envelope plasmid pHEF-VSVG (provided by the American National Institute of Health (NIH) aidsgene project) and the backbone plasmid pSG 3. delta. env. cmvFLUC (see Nie J, et al. Sci Rep.2017 Feb 20; 7:42769.) were CO-transfected into 293T cells at a mass ratio of 1:2 using the transfection reagent Lipofectamine 2000, the medium was changed about 4 to 6 hours after transfection, and then placed in a 37 ℃ 5% CO2 incubator for about 48 hours. The transfected cell culture medium supernatant was collected, centrifuged, cell debris removed, filtered through a 0.45 μm filter, and dispensed into 1.5mL centrifuge tubes and stored in a-80 ℃ freezer for use.
1.3 determination of pseudovirus titre
a) The split-frozen pseudovirus is taken out from a refrigerator at minus 80 ℃ and thawed in a room temperature water bath, 5 times of serial dilution is carried out in a 96-well plate, 11 gradients are carried out, 4 multiple wells are carried out, the final volume is 100 mu L, and the last column is taken as a cell control.
b) Taking 293T cells (with a confluence rate of more than 80%) prepared in an incubator in advance, taking a T75 incubator as an example, sucking and removing a culture medium in a bottle, adding 5ml of 1 XPBS to clean the cells, sucking and removing 1 XPBS, adding 2.5ml of 0.25% pancreatin-EDTA diluted by 5 times, immersing the cells for digestion for 45 seconds, sucking and removing pancreatin, placing the cells in the incubator for standing for 4 minutes until the cells fall off, adding 10ml of DMEM culture medium to neutralize the pancreatin, blowing and uniformly mixing, counting the cells, diluting the cells to 5 × 10 by using the DMEM complete culture medium5One per ml. Add 100. mu.L of each well, i.e., 5X 104And putting the 96-well plate into an incubator for culturing for 48 hours.
c) After 48h, the 96-well plate was removed from the cell incubator, 100. mu.L of the supernatant was aspirated from the sample well, and 100. mu.L of Bright-Glo luciferase assay reagent equilibrated to room temperature was added and reacted for 2min in the absence of light.
d) After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction hole for 3-5 times by using a multi-channel pipette to fully lyse the cells, sucking 100 mu l of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, placing the plate in a chemiluminescence detector, and reading the luminescence value by using a BrighGlo program.
e) The TCID50 of the pseudovirus is calculated by a Reed-Muench method by taking the luminous value which is 3 times higher than that of the cell control hole as the cut off value, and the specific steps are as follows:
calculating the number of positive wells (a) and the number of negative wells (b) of each virus dilution;
calculating the cumulative number of positive and negative wells: accumulating the positive hole accumulation number from bottom to top (c), and accumulating the negative hole accumulation number from top to bottom (d);
calculating the percentage of positive holes: a ratio of (c)/[ (c) + (d) ] × 100;
fourthly, calculating the distance proportion: distance ratio (percentage of positivity greater than 50% -50)/(percentage of positivity greater than 50% -percentage of positivity less than 50%);
the logarithm of TCID50 is the logarithm of the highest dilution factor of the percentage of positive greater than 50% + the distance ratio x the logarithm of the dilution factor.
Example 2 evaluation of inhibitory Activity of RV-07 on rabies pseudovirus
In this example, RV-07 (national Standard for Clofacial research, supplied by the national institute for food and drug testing, Standard substance for Chinese) was evaluated for rabies virus inhibitory activity by using the rabies pseudovirus as described above.
Since the pseudovirus backbone plasmid pSG3 delta env. cmvFluc used in the present invention is derived from HIV, in order to exclude the possibility that the drug inhibits the pseudovirus by preventing the reverse transcription process of the backbone HIV, the present invention uses a rabies pseudovirus primary screening drug and simultaneously uses a chimeric pseudovirus containing Vesicular Stomatitis Virus (VSV) membrane protein for secondary screening to exclude the false positive. The ratio of 50% inhibitory concentration (IC50) of the drug to the VSV membrane protein chimeric pseudovirus and rabies virus membrane protein chimeric pseudovirus was calculated, and when this value was greater than 3, the possibility of excluding the drug from inhibiting the pseudovirus by preventing the reverse transcription process of the framework HIV was judged. The specific experimental steps are as follows:
a) RV-07 was dissolved to 30mmol/L using DMSO.
b) RV-07 was serially diluted 3-fold in a 96-well plate starting from 150-fold dilutions, 8 dilutions, 2 duplicate wells, final volume of 100 μ L, and final first and second columns for cell control (medium and cells only) and virus control (virus and cells only), respectively.
c) Diluting rabies pseudovirus or VSV pseudovirus to 4000TCID50/ml, adding 50 μ L per well, i.e. 200TCID50 per well, placing 96-well plate at 37 deg.C, 5% CO2And culturing for 1 h.
d) When the incubation time is half an hour, the 293T cells prepared in the incubator in advance (the confluency rate is more than 80%) are taken out, and the cells are treated by the pancreatin. Cells were diluted to 5X 10 with DMEM complete medium5One per ml.
e) When the incubation time was up to 1 hour, 100. mu.l of 5X 10 was added to each well of a 96-well plate5Cells per ml, 5X 10 cells per well4Respectively, putting a 96-well plate into an incubator at 37 ℃ and 5% CO2Culturing for 48 h.
f) After 48h, the 96-well plate was removed from the cell culture chamber, 150. mu.l of the supernatant was aspirated from each well using a multi-channel pipette, and then 100. mu.l of Bright-GloTM luciferase assay reagent was added and reacted at room temperature in the dark for 2 min.
g) After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction hole for 3-5 times by using a multi-channel pipette to fully lyse the cells, sucking 100 mu l of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, placing the plate in a chemiluminescence detector, and reading the luminescence value by using a BrighGlo program.
h) Calculating the inhibition rate of the drug, wherein the inhibition rate is [1- (the mean value of the luminous intensity of the sample group-the mean value of the luminous intensity of the cell control wells)/(the mean value of the luminous intensity of the virus control wells-the mean value of the luminous intensity of the cell control wells) ] × 100%; and a dose-effect curve is drawn by taking the concentration of the drug as the abscissa and the inhibition rate as the ordinate.
i) From the inhibition rate curve of the drug, ID50 and IC50 of the drug were calculated using the following formulas.
Drug ID50 ═ lg (1/dilution of drug at a fluorescence value rate of less than 50%) + (50-percent fluorescence value from the drug at a fluorescence value rate of less than 50%)/(percent fluorescence value from the drug at a fluorescence value rate of greater than 50% > -percent fluorescence value from the drug at a fluorescence value rate of less than 50% >) × lg (drug dilution factor).
Drug IC50(μmol/L) 30000/drug ID 50.
The drug inhibition curves are shown in figure 1, and the IC50 of RV-07 to rabies pseudovirus (RV) is 1.7 mu M and the IC50 to VSV pseudovirus is 10.5 mu M, and the ratio of the two is obviously more than 3. The results show that RV-07 has a remarkable inhibitory effect on rabies pseudoviruses, and interferes with the infection of the rabies pseudoviruses on cells by inhibiting the combination of rabies virus membrane proteins and target cells.
Example 3 evaluation of the cytotoxicity of RV-07
In this example, the effect of RV-07 (national chloride-method standard, provided by the national institute for food and drug assay standard) on cell activity was evaluated by ATP quantification according to the following detection principle:
ATP is an indicator of viable cell metabolism using CellTiter-
The cell activity detection kit by the luminescence method detects the number of living cells in a culture after the action of a medicament and cells by carrying out quantitative determination on ATP, the luminescence signal generated by cell lysis is in direct proportion to the amount of ATP existing, and the amount of ATP is in direct proportion to the number of cells in the culture. The 50% cytotoxic concentration of the drug (CC50) was calculated by the effect of the drug on the ATP luminescence signal in the cells. The ratio of the 50% cytotoxicity concentration (CC50) of the drug to the IC50 of the drug to the target virus (namely, the therapeutic index SI ═ CC50/IC50) is calculated, and when the therapeutic index is more than 3, the drug is judged to be a positive drug for inhibiting the rabies virus, namely, the drug has obvious virus inhibition activity and no obvious cytotoxicity. The specific experimental steps are as follows:
a) RV-07 was serially diluted 3-fold in 96-well plates starting from 150-fold dilutions, 8 dilutions, 2 duplicate wells, final volume of 100 μ L, and negative controls (medium only, no cells) and cell controls (medium and cells only) in the last first and second columns, respectively.
b) Cell control and sample wells 50. mu.L of medium was added to each well, and 96-well plates were placed at 37 ℃ in 5% CO2And culturing for 1 h.
c) 293T cells prepared in advance in the incubator (the confluency was 80% or more) were removed, and cells were treated with trypsin as described above. Cells were diluted to 5X 10 with DMEM complete medium5One per ml. Add 100. mu.l of complete medium to the first column and 100. mu.l of diluted to 5X 10 per well to the remaining wells5293T cells at a density of 5X 10 cells per well4Respectively, putting a 96-well plate into an incubator at 37 ℃ and 5% CO2Culturing for 48 h.
d) After 48h, the 96 well plate was removed from the cell culture chamber, 150. mu.l of supernatant was aspirated from each well using a multi-channel pipette, and 100. mu.l of CellTiter-
And (5) detecting the reagent, and reacting for 10min at room temperature in a dark place.
e) After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction hole for 3-5 times by using a multi-channel pipette to fully lyse the cells, sucking 100 mu l of liquid from each hole, adding the liquid into a corresponding black 96-hole chemiluminescence detection plate, placing the plate in a chemiluminescence detector, and reading the luminescence value by using a CellTiter-Glo program.
f) Calculating the influence (inhibition rate) of RV-07 on the cell activity, wherein the inhibition rate is [1- (mean value of luminescence intensity of sample group-mean value of luminescence intensity of negative control well)/(mean value of luminescence intensity of cell control well-mean value of luminescence intensity of negative control well) ] × 100%; and a dose-effect curve is drawn by taking the concentration of the drug as the abscissa and the inhibition rate as the ordinate.
g) From the above inhibition curves, the 50% cytotoxic concentration of the drug (CC50) was calculated with reference to the formula described in example 2.
The results are shown in FIG. 2, where RV-07 has a CC50 for cells greater than 200. mu.M, indicating that RV-07 is not toxic to cells below 200. mu.M; also, the ratio of the CC50 value for RV-07 to its IC50 for rabies virus (i.e., therapeutic index SI ═ CC50/IC50) is significantly greater than 3. The results further show that RV-07 has obvious rabies virus inhibiting activity and no obvious cytotoxicity.
Example 4 evaluation of neutralizing Activity of RV-07 against rabies live Virus
In this example, live rabies virus was used to evaluate the neutralizing activity of RV-07 (national chloride Faqiming Standard, supplied by the Chinese food and drug assay institute Standard) on rabies virus.
With reference to FFIT procedures, 30mM RV-07 was diluted 150-fold and then serially diluted 3-fold for a total of 8 dilutions (200. mu.M, 66.7. mu.M, 22.2. mu.M, 7.4. mu.M, 2.5. mu.M, 0.8. mu.M, 0.3. mu.M, 0.1. mu.M), and RV-07 and 50. mu.l of rabies virus CVS strain (stored in the institute of food and drug assay in China) (6.25X 10. mu.M)5Fluorescence titer/ml) for 1 hour, and incubating the rabies virus CVS strain with PBS and DMSO for 1 hour respectively in the PBS and DMSO control groups; then, BSR cells (for the establishment and preservation of China food and drug testing institute) were added to each well, mixed and inoculated, and cell culture was continued at 37 ℃ for 24 hr. After the cell culture is finished, cell culture supernatant is discarded, PBS is washed for 1 time to remove residual virus in the cell supernatant as much as possible, acetone is used for fixing, and virus infection conditions in cells are observed through fluorescent staining.
The results are shown in fig. 3, where both the PBS and DMSO controls were strongly fluorescence positive, indicating that the virus infected BSR cells and propagated. The RV-07 drug group has obviously reduced fluorescence intensity, which shows that the RV-07 drug group has obvious effect of neutralizing the rabies virus CVS strain. Meanwhile, with the dilution of RV-07 (from left to right in the figure, the concentration of the drug is reduced in sequence), the fluorescence staining intensity is gradually enhanced, and the dosage relation of the RV-07 neutralizing rabies virus activity is shown. In the first three wells (drug concentration is 200. mu.M, 66.7. mu.M and 22.2. mu.M in sequence), the fluorescent staining is weak, which indicates that the virus is neutralized by the drug and loses the cell infection capacity; and the drug in the first three holes has no influence on the cells through microscopic observation. By the method shown in example 2, the IC50 of RV-07 to rabies virus CVS strain was calculated to be 34.5. mu.M (FIG. 4).
The results show that RV-07 has obvious neutralizing activity on the rabies live virus.
Example 5 evaluation of RV-07 in vivo anti-rabies Virus Activity
In this example, RV-07 was examined for anti-rabies virus activity in vivo using golden hamster after challenge with rabies virus as a model.
Golden yellow hamster 6-7 weeks old (provided by the animal resources center of the Chinese food and drug assay institute), was intramuscularly treated with standard challenge strain CVS, with an amount of challenge of 3.8LD50/ml, as well as a Control group (Control) and 4 experimental groups (Study), wherein the experimental groups included two doses (50. mu.l and 15. mu.l) of RV-07 oral treatment group and two doses (50. mu.l and 15. mu.l) of RV-07 intramuscular injection group. The results are shown in FIG. 5, after virus challenge, the Control group (Control) developed symptoms of morbidity within 6-7 days, 2 died on day 8, 4 survived, 66.7% survival rate, and all died on day 9. RV-07 oral treatment group (drug uses finished drug provided by available pharmaceutical products, pill, suspension oil liquid containing red or red brown powder, specification of 50 mg/pill, and drug volume of about 300 μ L in each pill, converted to concentration of about 352.1mmol/L) compared with control group, onset symptom appears 1d later, administration of 50 μ L group (figure 5A) dies 2 on day 9, survival rate of 66.7%, death rate of 2 on day 10, onset and death are continued, 1 survives finally, survival rate after drug intervention is 16.7%; the 15 μ l group (fig. 5B) died 2 on day 9, with a survival rate of 66.7%, 2 on day 10, 1 on day 12, and 1 on day 15. RV-07 intramuscular injections (the drug used was a finished drug provided by available from available pharmaceutical Co., Ltd., and the drug was in the form of gelatin capsules containing a suspension oil of a red or reddish brown powder, 50 mg/capsule in a specification, and the volume of the drug in each gelatin capsule was about 300. mu.l, which was converted to a concentration of about 352.1mmol/L) into 50. mu.l groups (FIG. 5C), 3 died on day 8, 50% survival, 2 died on day 9, and 1 died on day 10; the drug was injected intramuscularly in the 15 μ l group (fig. 5D), 1 death on day 7, 50% survival, 3 deaths on day 8, and 1 death on day 9. The results show that in the rabies virus infection in-vivo model, RV-07 can obviously delay the morbidity and the death time of golden hamster, improve the survival rate of golden hamster infected with rabies virus, and can be used for preventing and/or treating rabies virus infection and diseases caused by rabies virus infection.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Claims (7)
1. Use of a compound having the formula shown below or a pharmaceutically acceptable salt thereof as the sole active ingredient for the preparation of a pharmaceutical composition for the prevention and/or treatment of rabies virus infection or a disease caused by rabies virus infection in a subject
2. The use of claim 1, wherein the disease caused by rabies virus infection is rabies.
3. The use according to claim 1, wherein the pharmaceutical composition comprises a prophylactically or therapeutically effective amount of a compound as defined in claim 1.
4. The use of claim 1, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent having anti-rabies virus and/or anti-rabies activity.
5. The use of claim 4, wherein the additional pharmaceutically active agent is selected from rabies vaccine, anti-rabies serum or anti-rabies immunoglobulin.
6. The use of claim 5, wherein the anti-rabies immunoglobulin is an anti-rabies human immunoglobulin.
7. The use of claim 1, wherein the subject is a human.
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| US7201923B1 (en) * | 1998-03-23 | 2007-04-10 | General Mills, Inc. | Encapsulation of sensitive liquid components into a matrix to obtain discrete shelf-stable particles |
| WO2016063134A1 (en) * | 2014-10-24 | 2016-04-28 | Redhill Biopharma Ltd. | Therapy for inhibition of single-stranded rna virus replication |
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