Application of diphenylurea compounds in the preparation of anti-toxoplasmosis drugs

Abstract

The invention discloses the application of diphenylurea compounds in the preparation of anti-toxoplasmosis drugs. The present invention obtains 7 compounds that can effectively inhibit the growth of Toxoplasma through the in vitro anti-toxoplasmosis research on imidazole phenylurea and diphenylurea compounds, and has a high in vitro insect inhibition rate for Toxoplasma, can significantly inhibit the formation of parasite plaques, and well inhibit the proliferation of Toxoplasma; wherein diphenylurea compounds: 1,3-bis (3-fluorophenyl) urea, 1-(3-bromophenyl)-3-(4-chlorophenyl) urea, 1-(3-chlorophenyl)-3-(4-chlorophenyl) urea, have less cytotoxicity, can also be used to treat toxoplasmosis infected in mice, significantly inhibit Toxoplasma from reproducing in vivo, and will not affect the growth of mice, have a significant insect inhibition effect, and can be used to prepare and develop more anti-toxoplasmosis drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of diphenylurea compounds in the preparation of anti-toxoplasmosis drugs. Background Art

[0002] Toxoplasma gondii is an important zoonotic parasite that can infect almost all warm-blooded animals, including humans, and invade all nucleated cells. Toxoplasma infection can cause toxoplasmic eye disease and toxoplasmic encephalopathy, leading to miscarriage, stillbirth, fetal malformation, etc. in pregnant women or pregnant livestock. Most people with normal immunity can effectively control the proliferation of Toxoplasma through their own immune system after infection, so there are no obvious clinical symptoms. However, it is a serious hazard to immunocompromised individuals, such as HIV-infected patients and immunosuppressed organ transplant patients, or congenitally infected fetuses, which can significantly increase morbidity and mortality. In addition, Toxoplasma infection seriously endangers the healthy breeding of livestock and poultry, causing huge economic losses.

[0003] Toxoplasma has a wide range of hosts and multiple transmission routes. The life history of Toxoplasma is very complex. Its final host is a cat or a feline, and humans, pigs, cattle, sheep, etc. are its intermediate hosts. In the intermediate host, Toxoplasma can be transformed from rapidly proliferating tachyzoites to slowly proliferating bradyzoites under the host's immune pressure, forming tissue cysts that exist in the muscles or brain for a long time. Traditional anti-Toxoplasma drugs such as pyrimethamine and sulfadiazine are only effective against acute Toxoplasma infection, ineffective against chronic infection, and also have certain toxic side effects. Long-term use may cause the parasite to develop drug resistance. In addition, there are some alternative therapy drugs, such as clindamycin, atovaquone, etc., which also have the disadvantage of poor efficacy. Therefore, it is of great significance to find highly effective and low-toxic anti-Toxoplasma drugs.

[0004] Imidazolamide, also known as imipenem and imidocarb, was approved by the U.S. FDA in 1978 for the treatment of animal babesiosis. It is currently used in dogs for anti-toxoplasmosis treatment. However, since imidazolamide is not easily metabolized in animals, it is easily enriched in the kidneys and livers, and exists in the form of drug prototypes for a long time, causing hepato-renal toxicity, which to some extent hinders the scope of use of the drug. Therefore, it is of great practical significance to improve the safety of anti-toxoplasmosis drugs and develop more new drugs that are safe, non-toxic, and have no side effects for the treatment or prevention of toxoplasmosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing anti-toxoplasmosis drugs and provide the application of diphenylurea compounds in the preparation of anti-toxoplasmosis drugs.

[0006] The purpose of the present invention is to provide application of diphenylurea compounds in preparing anti-Toxoplasma drugs.

[0007] Another object of the present invention is to provide a preparation for inhibiting the growth of Toxoplasma gondii.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention studies the in vitro anti-Toxoplasma effect of imidazole phenylurea and diphenyl urea compounds, showing that the efficacy of imidazole phenylurea for the treatment of Toxoplasma is poor. Thirteen diphenyl urea compounds are obtained by chemical synthesis, and seven preferred compounds with good insect inhibition effect are discovered. The insect inhibition rate of the compounds on Toxoplasma is greater than 50%, and the in vitro replication of Toxoplasma can be well inhibited. Through in vitro insect inhibition experiments and cytotoxicity studies, three diphenyl urea compounds with low or no toxicity and high efficiency in inhibiting the growth of Toxoplasma are further obtained. The invention discloses a novel novel 1-(3-bromophenyl)-3-(4-chlorophenyl) urea, 1-(3-bromophenyl)-3-(4-chlorophenyl) urea, and 1-(3-chlorophenyl)-3-(4-chlorophenyl) urea, which can significantly inhibit the formation of parasite plaques and effectively inhibit the in vitro growth of Toxoplasma gondii. The invention can be further used to treat mice infected with Toxoplasma gondii, inhibit the reproduction of Toxoplasma gondii in mice, and can be used to treat toxoplasmosis in mice without affecting the growth of mice. The invention has a significant insect-inhibiting effect, can be used to prepare and develop more anti-Toxoplasma drugs, and has a good application prospect in the research and development of drugs for preparing, treating or preventing toxoplasmosis.

[0010] The structural formula of the diphenylurea compound provided by the present invention is as follows:

[0011]

[0012] Among them, R1 is selected from hydrogen, trifluoromethyl, cyano or halogen; R2 is selected from hydrogen or halogen; R3 is selected from hydrogen, trifluoromethyl, cyano or halogen; R4 is selected from hydrogen or halogen.

[0013] Therefore, the present invention provides the use of diphenylurea compounds in preparing preparations for inhibiting the growth or reproduction of Toxoplasma gondii.

[0014] Preferably, the formulation is used for inhibiting Toxoplasma gondii in vitro.

[0015] Preferably, the diphenylurea compound is selected from:

[0016] One or more of .

[0017] The invention provides application of diphenylurea compounds in preparing medicines for preventing Toxoplasma gondii infection.

[0018] The invention provides application of diphenylurea compounds in preparing medicines for treating toxoplasmosis.

[0019] Preferably, the diphenylurea compound is selected from: One or more of .

[0020] Preferably, the drug is capable of inhibiting the replication of Toxoplasma gondii tachyzoites.

[0021] Preferably, the drug is capable of inhibiting the growth of Toxoplasma gondii.

[0022] Preferably, the drug can inhibit the reproduction of Toxoplasma gondii in the body.

[0023] Furthermore, the drug comprises a diphenylurea compound or a pharmaceutically acceptable salt, hydrate or a combination thereof and excipients.

[0024] Furthermore, the drug is in the form of tablets, capsules, oral liquid preparations, sprays or injections.

[0025] The present invention has the following beneficial effects:

[0026] The invention provides application of diphenylurea compounds in preparing anti-toxoplasmosis drugs. The invention discovers 7 preferred compounds with good insect inhibition effect by chemical synthesis method, and the in vitro insect inhibition rate of Toxoplasma is greater than 50%, and the in vitro replication of Toxoplasma can be well inhibited. Through in vitro insect inhibition experiments and cytotoxicity research, three diphenylurea compounds with low toxicity or no toxicity and high efficiency in inhibiting the growth of Toxoplasma are further obtained, namely 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea and 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea, which can significantly inhibit the formation of parasite plaques and effectively inhibit the in vitro growth of Toxoplasma. At the same time, the diphenylurea compounds can also inhibit the reproduction of Toxoplasma in mice, and are used for treating toxoplasmosis infected mice without affecting the growth of mice. The compounds have significant therapeutic effect, can be used for preparing and developing more anti-toxoplasmosis drugs, and have good application prospects in the research and development of drugs for preparing, treating or preventing toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a preliminary screening result of the insect inhibition rate of different diphenylurea compounds.

[0028] Figure 2 This is a diagram showing the effect of diphenylurea compounds on the plaque formation of Toxoplasma gondii in vitro.

[0029] Figure 3 This is the statistical result of the effect of diphenylurea compounds on the plaque area of ​​Toxoplasma gondii.

[0030] Figure 4 This is a diagram showing the inhibitory effect of diphenylurea compounds on the proliferation of Toxoplasma gondii in mice.

[0031] Figure 5 This is a statistical chart of the inhibitory effect of diphenylurea compounds on the proliferation of Toxoplasma gondii in mice. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Example 1 Preliminary screening of anti-toxoplasma urea compounds

[0035] In the early stage of the present invention, different concentrations of imidazolinone were tested for in vitro insecticidal activity, and the EC values ​​of imidazolinone were determined. 50 is 1604 μM, due to the EC 50 The larger the value, the worse the efficacy, indicating that the inhibitory effect of imidazole phenyl urea on Toxoplasma gondii is not ideal and is not suitable for the treatment of toxoplasmosis. Thus, 13 diphenyl urea compounds (Table 1) were synthesized by chemical synthesis method. The specific compound synthesis method is referred to: Cheng Wang, Tian Hu, Jiayu Lu, et al. Convenient Diaryl Ureas as Promising Anti-pseudo-allergic Agents. [J]. Journal of Medicinal Chemistry, 2022, 65 (15): 10626-10637. doi: 10.1021 / acs.jmedchem.2c00846. The synthesis route is as follows:

[0036]

[0037] The compounds in Table 1 are all synthesized from aromatic amines in the presence of triphosgene by ’ -R4 ’ ) by introducing trifluoromethyl, hydroxyl, amidino, cyano, hydroxymethyl, halogen (F, Cl, Br) groups, 13 diphenylurea compounds were synthesized and characterized by high performance liquid chromatography (HPLC), 1 H-NMR, 13 The target compound was fully characterized by C-NMR and high-resolution mass spectrometry (HRMS), and the characterization results were consistent with the synthesized structure.

[0038] For example, the specific synthesis steps of compound No. 1 are as follows: 0.4 mol of bis(trichloromethyl) carbonate (BTC) is dissolved in anhydrous dichloromethane and stirred at 0°C for 5 min. 1 mol of substituted aniline and 1.2 mol of triethylamine are slowly added dropwise in sequence and stirred for 15 min respectively. Then a mixed solution of 1 mol of substituted aniline and 1.2 mol of triethylamine is added dropwise to the reaction solution and stirred for another 20 min. After the reaction, the solution is washed with saturated NaHCO3 and NaCl and dried over anhydrous sodium sulfate. The solvent is removed under vacuum to obtain a white residue, which is separated by chromatography to obtain a white powder product. After characterization, it can be seen that the corresponding compound has been successfully prepared by the above method.

[0039] In addition, other diphenylurea compounds in Table 1 can be prepared by the above method, so they are not characterized accordingly, but it does not affect the reaction and the acquisition of the target product. The structural formulas and names of 13 diphenylurea compounds prepared are shown in Table 1 below.

[0040] Table 1 Structural formula of diphenylurea compounds and their insect inhibition rate

[0041]

[0042]

[0043] The 13 diphenylurea compounds in Table 1 were tested for their in vitro anti-insect activity, and preliminary screening was performed in vitro at a concentration of 10 μM. The specific determination method is as follows:

[0044] (1) Human foreskin fibroblasts (HFF) (ATCC, Manassas, VA, USA) were used to culture the Toxoplasma RH strain (from Huazhong Agricultural University) in vitro, and when the parasites had large intraparasitic vacuoles, the parasites were lysed and released;

[0045] (2) The insect strain was inoculated on a newly grown 24-well plate with HFF cells and allowed to invade the cells for 1 h at 37°C and 5% CO2.

[0046] (3) Wash the uninvaded parasites with PBS, add the culture medium containing 10 μM compound to each well of the 24-well plate, and continue to culture the parasites at 37°C and 5% CO2 for 16 h;

[0047] (4) After washing three times with PBS, the cells were fixed with 4% paraformaldehyde at 37°C for 20 min and washed three times with PBS;

[0048] (5) Add rabbit anti-Toxoplasma gondii TgALD (1,6-bisphosphate fructose aldolase, prepared in our laboratory) polyclonal antibody and incubate for 30 min, then wash three times with PBS;

[0049] (6) The cells were permeabilized with 0.1% Triton X-100 for 20 min, washed three times with PBS, and then blocked with 10% FBS at 37°C for 2 h;

[0050] (7) Mouse anti-Toxoplasma positive serum (prepared in our laboratory) was added, and the 24-well plate was incubated in a 37°C incubator for 30 min, and then washed three times with PBS;

[0051] (8) Add Alexa Fluor 594-conjugated goat anti-rabbit (Cell Signaling Technology) and Alexa Fluor 488-conjugated goat anti-mouse (Cell Signalling Technology)) and 1:1000 diluted Hoechst (to label cell nuclei, Hoechst AG, Germany), then incubate in the dark for 30 min and wash three times with PBS;

[0052] (9) The slides were sealed and observed under an inverted fluorescence microscope. The number of vesicles containing 1, 2, 4, 8, or 16 or more parasites was recorded and counted. At least 100 vesicles were counted in each group. The experiment was repeated at least 3 times. The inhibition rate of parasites was obtained by comparing with the negative control (DMEM culture medium containing 2% FBS was used as the negative control). The inhibition rate of Toxoplasma gondii by different compounds was calculated using Graphpad Prism 8.

[0053] The statistical results of insect inhibition rates of different diphenylurea compounds are as follows: Figure 1 As shown in Table 1, 7 compounds: 3CF3-phenylurea, 3-CNphenylurea, 3-Clphenylurea, 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea, 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea have good inhibitory effects on Toxoplasma gondii, and the inhibition rate is greater than 50%; at the same time, 6 diphenylurea compounds have poor inhibitory effects on Toxoplasma gondii, or even cannot inhibit Toxoplasma gondii.

[0054] Example 2 In vitro insecticidal effects of candidate diphenylurea compounds

[0055] 1. Effects of diphenylurea compounds on tachyzoite replication in vitro

[0056] Determination of the EC of the 7 diphenylurea compounds screened in Example 1 50 The specific steps are as follows:

[0057] (1) Using HFF cells to culture the RH strain in vitro, when the parasite has a large parasite vesicle, the parasite is lysed and released;

[0058] (2) The insect strain was inoculated on a 24-well plate slide with grown HFF cells and allowed to invade the cells for 1 h at 37°C and 5% CO2.

[0059] (3) Wash the uninvaded insect strains with PBS, add culture medium containing different concentrations of diphenylurea compounds to the 24-well plate, and continue to culture the insects at 37°C and 5% CO2 for 16 h;

[0060] (4) After washing three times with PBS, the cells were fixed with 4% paraformaldehyde at 37°C for 20 min and washed three times with PBS;

[0061] (5) Add rabbit anti-Toxoplasma gondii TgALD polyclonal antibody and incubate for 30 min, then wash three times with PBS;

[0062] (6) Permeabilize cells with 0.1% Triton X-100 for 20 min, wash three times with PBS, and block with 10% FBS at 37°C for 2 h;

[0063] (7) Add mouse anti-Toxoplasma positive serum, incubate the 24-well plate in a 37°C incubator for 30 min, and wash three times with PBS;

[0064] (8) Add goat anti-rabbit 594 secondary antibody, goat anti-mouse 488 secondary antibody and Hoechst (1:1000 dilution), then incubate in the dark for 30 min and wash three times with PBS;

[0065] (9) The slides were sealed and observed under an inverted fluorescence microscope. The number of vesicles with 1, 2, 4, 8, or 16 or more parasites was recorded and counted. At least 100 vesicles were counted in each group. The experiment was repeated at least 3 times. The inhibition rate of parasites was calculated by comparing with the negative control. The EC value of the derivatives was calculated using Graphpad Prism 8. 50 .

[0066] The results are shown in Table 2. Among the seven diphenylurea compounds, 3CF3-phenylurea, 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea, 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea and 1-(3-bromophenyl)-3-(4-fluorophenyl)urea have better efficacy. 50 The EC values ​​of the other two compounds were 1.79μM, 1.67μM, 1.44μM, 1.35μM, and 2.33μM, respectively, which could effectively inhibit the growth of Toxoplasma gondii. 50 Greater than 5μM.

[0067] Table 2 EC of diphenylurea compounds against Toxoplasma gondii 50 Determination

[0068]

[0069] 2. Cytotoxicity assay

[0070] The CCK8 kit (Biyuntian, Cat. No.: C0038) was used to determine the effects of candidate diphenylurea compounds: 3CF3-phenylurea, 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea, and 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea on the growth of HFF cells at different concentrations. The specific steps are as follows:

[0071] (1) HFF cells were seeded in 96-well culture plates (10 4 cells / well) and cultured at 37°C, 5% CO2 for 24 h;

[0072] (2) Each compound was diluted to different concentrations with DMEM medium containing 2% FBS, and 100 μL was added to each well, with 3 replicates; the control group (containing only cells and medium) and the blank group without cells were only added with DMEM medium containing 2% FBS;

[0073] (3) After incubation for 48 h, the wells were washed three times with PBS, 100 μL of 10% CCK8 solution prepared with serum-free medium was added to each well, and after incubation for 1 h, the absorbance was detected at a wavelength of 450 nm using a multifunctional microplate reader;

[0074] The toxicity ratio of the corresponding concentration of the derivative to the cells was taken as [1-(experimental group-blank group) / (control group-blank group)]*100%, and the results were imported into Graphpad Prism 8 to calculate the TC of the derivative. 50 .

[0075] The results are shown in Table 3 below, showing the TC values ​​of diphenylurea compounds 3CF3-phenylurea, 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea, and 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea. 50 The values ​​are: 14.94μM, 112.10μM, 63.62μM, 122.40μM; among them, 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea, which has the most insecticidal activity, has the least cytotoxicity, and the selectivity of inhibiting the growth of the parasite is also greater than 100 times, and the selectivity of the remaining diphenylurea compounds for inhibiting the growth of the parasite is also at least greater than 10 times. These results show that the inhibitory effect of the diphenylurea compounds provided by the present invention on the growth of Toxoplasma gondii is not caused by causing significant host cell toxicity.

[0076] Table 3 Preferred diphenylurea compounds TC 50 Determination

[0077]

[0078] 3. Effects of candidate diphenylurea compounds on in vitro plaque formation of Toxoplasma gondii

[0079] (1) Using HFF cells to culture the RH strain in vitro, when the strain has large parasite vacuoles, the parasites are lysed and released, and the freshly overflowed Toxoplasma tachyzoites are filtered and purified using a 3 μm filter membrane;

[0080] (2) The insect strains were inoculated on a 6-well plate with HFF cells (100 Tg / well, 3 replicates for each insect strain) and the EC 50 The 6-well plates were incubated with diphenylurea compounds of different concentrations; 5 μM pyrimethamine was set as a positive control; the negative control was 2% FBSDMEM medium without drug addition and given the same volume, and cultured at 37°C and 5% CO2 for 7 days;

[0081] (3) After 7 days, the 6-well plate was washed 1-2 times with PBS, and the cells were fixed with 4% paraformaldehyde at 37°C for 20 min and washed 1-2 times with PBS;

[0082] (4) Stain with 0.1% crystal violet for 20 min, wash with PBS and dry at room temperature;

[0083] (5) Scan the plaque with a scanner and measure the area of ​​the plaque.

[0084] The results of the effects of diphenylurea compounds on the growth of Toxoplasma gondii in vitro are as follows Figure 2 and Figure 3 As shown, compared with the control group without compound addition, the RH strain cultured with 1,3-bis(3-fluorophenyl)urea, 1-(3-bromophenyl)-3-(4-chlorophenyl)urea, and 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea could not form visualized plaques, indicating that these compounds have low cytotoxicity and can significantly inhibit the in vitro growth of Toxoplasma gondii.

[0085] Example 3 In vivo insecticidal effects of candidate diphenylurea compounds

[0086] The diphenylurea compound 1-(3-chlorophenyl)-3-(4-chlorophenyl)urea which can significantly inhibit the in vitro growth of Toxoplasma gondii in Example 2 was selected for in vivo insect inhibition effect determination. Mice infected with Toxoplasma gondii (Ruige Biotechnology Co., Ltd.) were treated, and the RH-Luc strain (from Huazhong Agricultural University) was inoculated in the mice. After 4 hours, the drug treatment was started (with pyrimethamine as the positive control, 20 mg / kg, the drug was dissolved in corn oil or 50% ethanol; the negative control was given an equal volume of water), and the drug was administered daily for 5 days before the in vivo imaging experiment. The specific steps are as follows:

[0087] (1) D-luciferin sodium salt (10 μL / g; 15 mg / mL) was injected intraperitoneally 20 min before the experiment and anesthetized;

[0088] (2) using a live imaging device to image the mouse in the abdominal position and collect luminescent images of luciferase expressed by parasites in the mouse;

[0089] (3) The data were analyzed using Living Image and then imported into Prism 8 for statistical analysis.

[0090] The results are as follows Figure 4 and Figure 5 As shown, compared with the untreated control group, the bioluminescence intensity of Toxoplasma gondii in the experimental group of mice administered with 1-(3-chlorophenyl)-3-(4-chlorophenyl) urea was significantly reduced, indicating that treatment with 1-(3-chlorophenyl)-3-(4-chlorophenyl) urea has a certain inhibitory effect on the reproduction of Toxoplasma gondii in mice, and will not affect the growth of mice, and can be used to prevent or treat toxoplasmosis.

[0091] In summary, the present invention has discovered 7 diphenylurea compounds that can inhibit Toxoplasma through the research and development of anti-Toxoplasma drugs. They can well inhibit the growth of Toxoplasma in vitro and can significantly inhibit the formation of parasite plaques; at the same time, they can also be used to inhibit the reproduction and growth of Toxoplasma in vivo, have low toxicity to cells, can be used to treat toxoplasmosis in mice, and will not affect the growth of mice. They have significant insect inhibitory effects and biosafety, and can be used to prepare and develop more anti-Toxoplasma drugs and inhibitors.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a diphenylurea compound in the preparation of a preparation for inhibiting the growth or reproduction of Toxoplasma gondii, characterized in that: The diphenylurea compound is selected from: , , , , , or One or more of .

2. Use of diphenylurea compounds in the preparation of drugs for preventing Toxoplasma gondii infection, characterized in that: The diphenylurea compound is selected from: , or One or more of .

3. Use of diphenylurea compounds in the preparation of drugs for treating toxoplasmosis, characterized in that: The diphenylurea compound is selected from: , or One or more of .

4. The use according to claim 2 or 3, characterized in that: The drug can inhibit the replication of Toxoplasma gondii tachyzoites.

5. The use according to claim 2 or 3, characterized in that: The drug can inhibit the growth of Toxoplasma gondii.

6. The use according to claim 2 or 3, characterized in that: The drug can inhibit the reproduction of Toxoplasma gondii in the body.

7. The use according to claim 2 or 3, characterized in that: The medicine comprises a diphenylurea compound or a pharmaceutically acceptable salt or an excipient thereof.

8. The use according to claim 2 or 3, characterized in that: The dosage form of the drug is tablet, capsule, oral liquid preparation, spray or injection.

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