Application of squaric acid compound in preparation of toxoplasmosis resisting medicine

Abstract

The invention discloses an application of squaric acid compounds in preparation of drugs for resisting toxoplasmosis. According to the novel application of the squaric acid compounds in resisting toxoplasma gondii infection provided by the invention, two preferable squaric acid compounds, namely F-squaric acid and 3-F-squaric acid, with good insect inhibition effects are explored through synthesis and insect inhibition experiments of the squaric acid compounds, the in-vitro insect inhibition rate of the squaric acid compounds on toxoplasma gondii is high, in-vitro replication of the toxoplasma gondii can be well inhibited, and the squaric acid compounds can be applied to prevention of toxoplasma gondii infection. The formation of polypide plaques is obviously inhibited, and the growth of toxoplasma gondii can be effectively inhibited; and the compound has good safety, does not have cytotoxicity, and belongs to a low-toxicity or non-toxic squaric acid compound. Meanwhile, the squaric acid compound provided by the invention also can inhibit the breeding of toxoplasma gondii in a mouse body and treat toxoplasmosis, has a remarkable treatment effect, does not influence the growth of the mouse, can resist toxoplasma gondii infection, can be used for preparing and developing more products for resisting toxoplasma gondii infection and medicines for treating toxoplasmosis, and has a wide application prospect. The method is of great significance to research and development of drugs for treating toxoplasmosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to the application of squaric acid compounds in the preparation of drugs for treating toxoplasmosis. Background Art

[0002] Toxoplasma gondii is an important zoonotic parasitic protozoan that can infect almost all warm-blooded animals including humans and invade all nucleated cells. Toxoplasma gondii has a wide host range and diverse transmission routes. The life cycle of Toxoplasma gondii is very complex, with its definitive host being cats or felids, and humans, pigs, cattle, sheep, etc. being its intermediate hosts. In intermediate hosts, under the immune pressure of the host, Toxoplasma gondii can transform from rapidly proliferating tachyzoites into slowly proliferating bradyzoites, forming tissue cysts that can exist in muscles or the brain for a long time. Most people with normal immunity can effectively control the proliferation of Toxoplasma gondii through their own immune systems and thus do not show obvious clinical symptoms. However, for individuals with low immune function, it can cause lymphadenopathy, central nervous system damage, and toxoplasmic ophthalmopathy, etc. Toxoplasma gondii infection can lead to miscarriage, stillbirth, fetal malformation, etc. in pregnant women or pregnant livestock; children and the elderly are prone to diseases such as meningitis and pneumonia after being infected with Toxoplasma gondii. Toxoplasma gondii infection in livestock such as pigs, cattle, and sheep can also cause reproductive disorders or miscarriage, seriously endangering the development of animal husbandry and causing huge economic losses.

[0003] Traditional anti-Toxoplasma gondii drugs such as pyrimethamine and sulfadiazine have good therapeutic effects on acute infections but are accompanied by varying degrees of toxic and side effects, and they are ineffective against chronic infections. If used for a long time, it is easy for the parasite to develop drug resistance. In addition, there are some alternative therapy drugs such as clindamycin and atovaquone, which although have certain therapeutic effects on chronic infections, still cannot eradicate the infection. Therefore, it is of great significance to find more highly effective and low-toxic anti-Toxoplasma gondii drugs.

[0004] Developing anti-Toxoplasma gondii drugs can also be achieved by developing natural products and synthesizing new drugs targeting drug targets. Repurposing old drugs has the advantages of low cost and significantly shortening the time for drug research and development. Imidocarb is a good anti-Babesia drug and has the potential for anti-parasitic effects. However, due to the fact that imidocarb is not easily metabolized in animals and is easily enriched in the kidneys and liver and exists in the form of the drug prototype for a long time, causing liver and kidney toxicity, to a certain extent, it hinders the scope of use of this drug. Therefore, it is extremely urgent to improve the safety of anti-Toxoplasma gondii drugs and develop more new drugs that can be used to treat or prevent toxoplasmosis and are safe, non-toxic, and have no side effects, which has very important practical significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of existing anti - toxoplasmosis drugs, such as side effects, drug resistance, and low safety, and to provide the application of squaric acid compounds in the preparation of anti - toxoplasmosis drugs.

[0006] The object of the present invention is to provide a new application of squaric acid compounds in anti - toxoplasmosis.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides the application of squaric acid compounds in the preparation of drugs for treating toxoplasmosis. The structural formula of the squaric acid compounds is as follows:

[0009] Among them, R1 - R4 are selected from hydrogen or halogen F.

[0010] The research of the present invention shows that squaric acid compounds with specific structures have the effect of inhibiting the growth of Toxoplasma gondii, with a high inhibition rate of Toxoplasma gondii. They can not only effectively inhibit the in - vitro replication of Toxoplasma gondii and inhibit its growth, but also significantly inhibit the formation of parasite plaques and the proliferation of Toxoplasma gondii. At the same time, the EC 50 of the compound is relatively low, having good drug efficacy and good anti - Toxoplasma potential. In addition, the squaric acid compounds provided by the present invention have good safety, with less toxicity to HFF cells, belonging to low - toxicity or non - toxic squaric acid compounds. Further, the in - vivo experimental results show that squaric acid compounds can inhibit the reproduction of Toxoplasma gondii in mice, have a certain therapeutic effect on mice infected with toxoplasmosis, and do not affect the normal growth of mice. The present invention provides a new application of specific squaric acid compounds in anti - toxoplasmosis, providing new ideas and methods for the preparation and development of more anti - toxoplasmosis drugs, and having good application prospects in the research and development of drugs for treating or preventing toxoplasmosis.

[0011] Therefore, the present invention provides the application of squaric acid compounds in the preparation of products for inhibiting the growth or reproduction of Toxoplasma gondii.

[0012] The present invention provides the application of squaric acid compounds in the preparation of products for anti - Toxoplasma infection.

[0013] Preferably, the squaric acid compound is:

[0014]

[0015] Furthermore, the product or drug can inhibit the growth of Toxoplasma gondii.

[0016] Furthermore, the product or drug can inhibit the proliferation of Toxoplasma gondii.

[0017] Furthermore, the product or drug can inhibit the replication of Toxoplasma gondii tachyzoites.

[0018] Furthermore, the product or drug can inhibit the formation of Toxoplasma plaques.

[0019] Preferably, the product further comprises squaric acid compounds or pharmaceutically acceptable salts, hydrates or combinations thereof, or excipients.

[0020] Preferably, the dosage form of the drug is tablets, capsules, oral liquid preparations, sprays or injections.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a new application of squaric acid compounds in the preparation of drugs for treating toxoplasmosis. Through the synthesis and anti-parasitic experiments of squaric acid compounds, two preferred squaric acid compounds with good anti-parasitic effects, namely p-F-squaric acid and 3-F-squaric acid, are discovered. They have a relatively high in vitro anti-Toxoplasma rate, can well inhibit the in vitro replication of Toxoplasma, significantly inhibit the formation of parasite plaques, and can effectively inhibit the in vitro growth of Toxoplasma. And they have good safety, no cytotoxicity, and belong to low-toxic or non-toxic squaric acid compounds. The EC 50 is relatively low, with good drug efficacy and good anti-Toxoplasma potential. At the same time, the squaric acid compounds provided by the present invention can also inhibit the reproduction of Toxoplasma in mice, treat toxoplasmosis, and do not affect the growth of mice. They have a significant therapeutic effect, can resist Toxoplasma infection, and can be used to prepare and develop more products for anti-Toxoplasma infection and drugs for treating toxoplasmosis, showing good application prospects for the research and development of drugs for treating or preventing toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a preliminary screening result diagram of the anti-parasitic rates of different squaric acid compounds.

[0024] Figure 2 It is a diagram of the formation of Toxoplasma plaques in vitro by squaric acid compounds.

[0025] Figure 3 It is a statistical result diagram of the plaque area of Toxoplasma by squaric acid compounds.

[0026] Figure 4 It is a result diagram of the virulence survival curve of Toxoplasma in mice by squaric acid compounds. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0028] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0029] Synthesis of Squaric Acid Compounds in Example 1

[0030] Referring to the prior art (Pfeifer, Lukas, et al, Hydrogen-Bonded Homoleptic Fluoride-Diarylurea Complexes: Structure, Reactivity, and Coordinating Power; Journal of the American Chemical Society (2016), 138(40), 13314-13325.), 3,4-diethoxycyclobutene-3-ene-1,2-dione reacts with amine compounds with different substituents and zinc trifluoromethanesulfonate in a mixed solution of an appropriate amount of toluene and N,N-dimethylformamide, and five squaric acid compounds are synthesized by chemical synthesis method. The synthesis route is as follows:

[0031]

[0032] By introducing trifluoromethyl, cyano, hydroxyl, and halogen (F) groups into R of amine compounds with different substituents, squaric acid compounds with different substituents can be synthesized. For example, the specific synthesis steps of Compound 5 are as follows: Add anhydrous toluene (38 mL), anhydrous DMF (2 mL), 3,4-diethoxycyclobut-3-ene-1,2-dione (5.11 g, 30.0 mmol), and zinc trifluoromethanesulfonate (2.18 g, 6.0 mmol) to a dry 100 mL round-bottom flask. The resulting suspension is stirred at room temperature, and then 4-fluoroaniline (6.67 g, 60.0 mmol) is added. The solution is heated to 100 °C and stirred for 18 hours, and at this time, a yellow precipitate is observed. The reaction mixture is cooled to room temperature, filtered, washed successively with methanol (3 * 50 mL) and ether (50 mL), and dried under high vacuum to obtain the desired product, which is a powdery, light yellow solid (8.65 g, 96% yield).

[0033] The following compounds are all prepared by the above method and are comprehensively characterized by 1 1H-NMR, and the characterization results are consistent with the synthetic structure.

[0034] Compound 1: 3,4-bis{[3-(trifluoromethyl)phenyl]amino}cyclobut-3-ene-1,2-dione. 11H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 2H), 7.90 (s, 2H), 7.61 (d, J = 8.0 Hz, 4H), 7.42 (d, J = 6.8 Hz, 2H).

[0035] Compound 2: 4-{[4-(Ethylamino)-2,3-dioxocyclobut-4-en-1-yl]amino}benzene-1-carbonitrile. 1 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.84 - 7.79 (m, 2H), 7.59 - 7.54 (m, 2H), 4.80 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 6.8 Hz, 3H).

[0036] Compound 3: 3,4-Bis[(4-fluorophenyl)amino]cyclobut-3-ene-1,2-dione. 1 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 2H), 7.53 - 7.43 (m, 4H), 7.22 (t, J = 8.8 Hz, 4H).

[0037] Compound 4: 3,4-Bis[(3-hydroxyphenyl)amino]cyclobut-3-ene-1,2-dione. 1 1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 2H), 9.60 (s, 2H), 7.15 (t, J = 8.0 Hz, 2H), 6.97 - 6.91 (m, 4H), 6.53 - 6.47 (m, 2H).

[0038] Compound 5: 3,4-Bis[(3-fluorophenyl)amino]cyclobut-3-ene-1,2-dione. 1 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 2H), 7.50 (dt, J = 11.2, 2.4 Hz, 2H), 7.42 (td, J = 8.2, 6.4 Hz, 2H), 7.19 (dd, J = 8.0, 2.4 Hz, 2H), 6.92 (td, J = 8.4, 2.5 Hz, 2H).

[0039] Insecticidal rate of squaric acid compounds in Example 2

[0040] Five squaric acid compounds (prepared in Example 1) were used at a concentration of 10 μM for in vitro anti-insect activity tests. The specific method was as follows:

[0041] (1) The human foreskin fibroblasts (HFF) (ATCC, Manassas, VA, USA) were used to culture the Toxoplasma gondii RH strain (obtained from Huazhong Agricultural University) in vitro. When the parasites had large parasitophorous vacuoles, the parasites were lysed and released.

[0042] (2) The parasite strain was inoculated onto the coverslips in a 24-well plate with confluent HFF cells and allowed to invade the cells for 1 h at 37 °C and 5% CO2.

[0043] (3) The non-invasive parasites were washed away with PBS, and the medium containing 10 μM of the compound was added to the 24-well plate, and the parasites were further cultured for 16 h at 37 °C and 5% CO2.

[0044] (4) The free parasites were washed away with PBS, and the coverslip cells were fixed with 4% paraformaldehyde for 20 min at 37 °C and washed 3 times with PBS.

[0045] (5) Rabbit anti-Toxoplasma gondii TgALD (fructose-bisphosphate aldolase, prepared and stored in our laboratory) polyclonal antibody was added and incubated for 30 min, and then washed 3 times with PBS.

[0046] (6) The cells were permeabilized with 0.1% Triton X-100 for 20 min, washed 3 times with PBS, and then blocked with 10% FBS for 2 h at 37 °C.

[0047] (7) Mouse anti-Toxoplasma gondii 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 3 times with PBS.

[0048] (8) Alexa Fluor 594-conjugated goat anti-rabbit (Cell Signalling Technology), Alexa Fluor 488-conjugated goat anti-mouse (Cell Signalling Technology) and Hoechst (labeling cell nuclei, Hoechst AG, Germany) diluted at 1:1000 were added, and then incubated in the dark for 30 min and washed 3 times with PBS.

[0049] (9) Mount the slides, observe, record and count the number of parasites in parasitophorous vacuoles under an inverted fluorescence microscope. The number of parasitophorous vacuoles with 1, 2, 4, 8, or 16 or more parasites was counted. At least 100 parasitophorous vacuoles were counted in each group, and the experiment was repeated at least 3 times independently. By comparing with the negative control (DMEM medium containing 2% FBS was used as the negative control), the inhibition rate of the parasites was obtained, and the inhibition rate of different compounds against Toxoplasma gondii was calculated using Graphpad Prism 8.

[0050] The statistical results of the insecticidal rates of different squaric acid compounds are as Figure 1 shown in Table 1, indicating that among the 5 compounds, F-squaric acid and 3-F-squaric acid have good inhibitory effects on Toxoplasma gondii, and the insecticidal rate of 3-F-squaric acid is greater than 65%. At the same time, the other 3 squaric acid compounds have poor inhibitory effects on Toxoplasma gondii, and 3-OH squaric acid cannot even inhibit Toxoplasma gondii.

[0051] Table 1 Structural formulas of squaric acid compounds and their insecticidal rates

[0052]

[0053]

[0054] Example 3 In vitro insecticidal effect of squaric acid compounds

[0055] 1. Effect of squaric acid compounds on the in vitro replication of tachyzoites

[0056] Determine the EC 50 of F-squaric acid and 3-F-squaric acid compounds with good inhibitory effects in Example 2. The specific method steps are as follows:

[0057] (1) Cultivate the Toxoplasma gondii RH strain in vitro using HFF cells. When the parasites have large parasitophorous vacuoles, lyse the parasites to release them.

[0058] (2) Inoculate the parasite strain on the coverslips of a 24-well plate with well-grown HFF cells and invade the cells at 37°C and 5% CO2 for 1 h.

[0059] (3) Wash away the non-invaded parasite strain with PBS, add media containing different concentrations of squaric acid compounds to the 24-well plate, and continue to culture the parasites at 37°C and 5% CO2 for 16 h.

[0060] (4) Wash away the free parasites with PBS, fix the coverslip cells with 4% paraformaldehyde at 37°C for 20 min, and wash 3 times with PBS.

[0061] (5) Add rabbit anti-Toxoplasma gondii TgALD polyclonal antibody and incubate for 30 min, then wash 3 times with PBS.

[0062] (6) Permeabilize the cells with 0.1% Triton X-100 for 20 min. After washing 3 times with PBS, block them with 10% FBS at 37 °C for 2 h;

[0063] (7) After washing 3 times with PBS, add mouse anti-Toxoplasma gondii positive serum to the 24-well plate, incubate it in an incubator at 37 °C for 30 min, and wash 3 times with PBS;

[0064] (8) Add goat anti-rabbit 594 secondary antibody, goat anti-mouse 488 secondary antibody and Hoechst (diluted 1:1000), then incubate in the dark for 30 min, and wash 3 times with PBS;

[0065] (9) Mount the slides, observe under an inverted fluorescence microscope, record and count the number of parasitophorous vacuoles with 1, 2, 4, 8, or greater than or equal to 16 parasites in each parasitophorous vacuole. At least 100 parasitophorous vacuoles are counted in each group, and the experiment is repeated at least 3 times separately. By comparing with the negative control, calculate the inhibition rate of parasites at different concentration gradients, and calculate the EC of the compound using Graphpad Prism 8 50 。

[0066] The results are shown in Table 2, indicating that the two squaric acid compounds have EC values for F-squaric acid and 3-F-squaric acid 50 of 5.69 μM and 0.997 μM respectively. The lower the EC 50 , the better the efficacy, and it can effectively inhibit the growth of Toxoplasma gondii.

[0067] Table 2 EC values of squaric acid compounds against Toxoplasma gondii 50

[0068]

[0069] 2. Cytotoxicity experiment

[0070] The above determination shows that 3-F-squaric acid has a better inhibitory effect. Further, the cytotoxicity of this compound was determined. The CCK8 kit (Beyotime, catalog number: C0038) was used to determine the effect of the squaric acid compound 3-F-squaric acid at different concentrations on the growth of HFF cells. The specific steps are as follows:

[0071] (1) Seed HFF cells into a 96-well culture plate at a density of 10 4 cells / well and culture them at 37 °C and 5% CO2 for 24 h;

[0072] (2) Dilute the squaric acid compounds to different concentrations with DMEM medium containing 2% FBS. Add 100 μL of the corresponding compound medium to each well and set up 3 replicate wells; use DMSO 2% FBS DMEM medium with the same volume as the compound added and the blank group without cells (only add DMEM medium containing 2% FBS) as the control group (only containing cells and medium), and also set up 3 replicate wells;

[0073] (3) After incubating for 48 h, wash 3 times with PBS. Add 100 μL of 10% CCK8 solution prepared with serum-free medium to each well. After incubating for 1 h, detect the absorbance at a wavelength of 450 nm using a multifunctional microplate reader;

[0074] (4) Use [1 - (experimental group - blank group) / (control group - blank group)] * 100% as the toxicity ratio of the corresponding concentration of the compound to the cells, calculate the relative toxicity ratio, and import the results into Graphpad Prism 8 to calculate the TC of the compound 50 .

[0075] The results are shown in Table 3 below, showing that the TC of the squaric acid compound 3-F-squaric acid 50 value is: 146.7 μM, with less toxicity to cells and a selectivity for inhibiting the growth of the worm body greater than 100 times, indicating that the inhibitory effect of the squaric acid compound 3-F-squaric acid on the growth of Toxoplasma gondii is not due to significant host cell toxicity.

[0076] 3. Effect of squaric acid compounds on plaque formation of Toxoplasma gondii in vitro

[0077] (1) Use HFF cells to culture the RH strain in vitro. When the strain has a large parasitophorous vacuole, lyse the worms to release them, and filter and purify the freshly overflowed tachyzoites of Toxoplasma gondii using a 3 μm filter membrane;

[0078] (2) Inoculate the RH strain (100 Tg / well, with 3 parallels for each strain) on a 6-well plate seeded with HFF cells, and incubate the seeded 6-well plate with 3-F-squaric acid at the EC 50 concentration; at the same time, set 5 μM pyrimethamine as the positive control; use 2% FBS DMEM medium without adding the compound and with the same volume as the negative control, and culture at 37 °C and 5% CO2 for 7 d;

[0079] (3) After 7 d, wash the 6-well plate 1 - 2 times with PBS, and fix the cells on the 6-well plate with 4% paraformaldehyde at 37 °C for 20 min, then wash 1 - 2 times with PBS;

[0080] (4) Stain with 0.1% crystal violet for 20 min, wash with PBS and air dry at room temperature;

[0081] (5) Scan the plaques with a scanner and measure the plaque area.

[0082] The statistical chart of plaque formation and plaque area of Toxoplasma gondii in vitro is as Figure 2 and Figure 3 shown. Compared with the control group, the RH strain cultured with 3-F-squaric acid could not form visible plaques, which affected the in vitro growth of Toxoplasma gondii, indicating that 3-F-squaric acid not only had low cytotoxicity but also could significantly inhibit the in vitro growth of Toxoplasma gondii.

[0083] Example 4 Inhibitory effect of squaric acid compounds on worms in vivo

[0084] The inhibitory effect of 3-F-squaric acid on worms in vivo was measured, and ICR mice infected with Toxoplasma gondii (purchased from Regene Biotechnology Co., Ltd.) were treated: The ME49 strain (derived from Anhui Agricultural University) was inoculated into mice at 10 4 / mouse. Four hours later, the first drug treatment was carried out (50 mg / kg 3-F-squaric acid, with pyrimethamine as the positive control, 20 mg / kg, and the compound was dissolved in 5% absolute ethanol + corn oil; the negative control was given the same volume of corn oil). The drug was administered daily. After 7 days, a virulence experiment was carried out to observe the death situation and weight change of the mice. The specific steps were as follows:

[0085] (1) Using serum-free DMEM solution as the diluent, inoculate mice at an infection dose of 10 4 tachyzoites / mouse, and intraperitoneally inject the tachyzoites of the ME49 strain to infect 7-week-old female ICR mice, with 8 mice inoculated in each group;

[0086] (2) After 7 days of infection, observe the death situation and weight change of the mice continuously for 23 days.

[0087] The detection results are as Figure 4 shown, showing that the treatment effect of the squaric acid compound 3-F-squaric acid can increase the survival rate of mice to 50%, and the compound does not affect the growth of mice, has a significant inhibitory effect on worms, and has an anti-Toxoplasma gondii infection effect. Although the in vivo treatment effect is not as good as that of pyrimethamine, its in vitro effect is basically the same as that of pyrimethamine. Considering that it is a compound with potential, it can be further optimized.

[0088] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of squaric acid compounds in the preparation of a product for inhibiting the growth or reproduction of Toxoplasma gondii, characterized in that, The structural formula of the squaric acid compound is as follows: Among them, R1 to R4 are selected from hydrogen or halogen F.

2. Application of squaric acid compounds in the preparation of products against Toxoplasma gondii infection, characterized in that, The structural formula of the squaric acid compound is as follows: Among them, R1 to R4 are selected from hydrogen or halogen F.

3. Use of squaric acid compounds in the preparation of drugs for treating toxoplasmosis, characterized in that, The structural formula of the squaric acid compound is as follows: Wherein, R1 to R4 are selected from hydrogen or halogen F.

4. The application according to any one of claims 1 to 3, characterized in that The squaric acid compound is as follows:

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

6. The application according to claim 2 or 3, characterized in that, The product or drug can inhibit the proliferation of Toxoplasma gondii.

7. The application according to claim 2 or 3, characterized in that The product or drug can inhibit the replication of Toxoplasma gondii tachyzoites.

8. The application according to claim 2 or 3, characterized in that, The product or drug can inhibit the formation of Toxoplasma gondii plaques.

9. The application according to claim 1 or 2, characterized in that, The product also contains a squaric acid compound or a pharmaceutically acceptable salt, hydrate or combination thereof, or an excipient.

10. The application according to claim 3, characterized in that, The dosage form of the drug is tablets, capsules, oral liquid preparations, sprays or injections.

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