CN110302199B - Application of tipranavir in preparation of medicine for inhibiting growth of toxoplasma gondii - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
- A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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Abstract
The invention relates to an application of tipranavir in preparation of a medicine for inhibiting the growth of toxoplasma gondii, belonging to the technical field of medicine preparation. The invention provides application of tipranavir in preparing a medicament for inhibiting the growth of toxoplasma gondii. The tipranavir can inhibit the growth of toxoplasma gondii.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to application of tipranavir in preparation of a medicine for inhibiting the growth of toxoplasma gondii.
Background
Toxoplasma is an intracellular parasitic protozoa, is a pathogen causing toxoplasmosis in both humans and animals, and is widely distributed in China and all over the world. Toxoplasmosis is not only one of the most common infectious diseases in humans, but is also one of the major lethal causes of immunosuppressed and immunodeficient people. In recent years, AIDS combined with Toxoplasma infection has become an increasingly serious public health problem worldwide.
Toxoplasma gondii infection is usually recessive infection, but when the immune function of a human body is low, the Toxoplasma gondii in the recessive infection stage is activated and continuously proliferated to seriously damage the organs of the human body. AIDS patients will have different clinical symptoms after being infected with toxoplasma, and when serious, the patients will die. Toxoplasma encephalopathy occurs in 20-47% of cases during HIV infection. The infection of toxoplasma by AIDS patients is usually treated by sulfadiazine drugs, however, with the appearance of HIV inhibiting drugs in the 90 s, the number of AIDS patients who are killed by the infection of toxoplasma is found to be obviously reduced in clinic. However, the effect of a few antiretroviral drugs on the inhibition of Toxoplasma gondii proliferation was evaluated. There are several distinct classes of HIV inhibitory drugs including protease inhibitors, integrase inhibitors, invasion inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. The selection of different types of HIV inhibiting drugs may affect the outcome of Toxoplasma gondii infection in AIDS infected individuals. However, it is not clear whether the HIV-inhibiting drugs used to date for the treatment of HIV infection have a direct inhibitory effect on the growth of Toxoplasma gondii, and the possible pharmacokinetic interactions between them and conventional anti-Toxoplasma gondii drugs (sulfadiazine and pyrimethamine).
Disclosure of Invention
The invention aims to provide application of tipranavir in preparing a medicament for inhibiting the growth of toxoplasma gondii. The tipranavir can inhibit the growth of toxoplasma gondii.
The invention provides application of a compound tipranavir shown as a formula I in preparation of a medicament for inhibiting the growth of toxoplasma gondii;
the invention also provides application of a composition containing the compounds of the tipranavir and the sulfadiazine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of a composition containing the compounds of the nelfinavir and the pyrimethamine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of a composition containing the compounds of tipranavir, sulfadiazine and pyrimethamine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of the compound tipranavir shown in the formula I in preparation of a medicine for preventing or treating toxoplasma gondii infection diseases.
The invention also provides application of a composition containing the compounds of the tipranavir and the sulfadiazine shown in the formula I in preparing a medicament for preventing or treating toxoplasma infection diseases.
The invention also provides application of a composition containing the compounds of the nelfinavir and the pyrimethamine shown in the formula I in preparing a medicament for preventing or treating toxoplasma gondii infection diseases.
The invention also provides application of a composition containing the compounds of tipranavir, sulfadiazine and pyrimethamine shown in the formula I in preparing a medicament for preventing or treating toxoplasma infection diseases.
The invention also provides a medicament for preventing or treating toxoplasma infection diseases, which comprises tipranavir and one or two of the following compounds: sulfadiazine and pyrimethamine.
The invention provides a method for preparing tipranavir for inhibiting bowApplication in medicine for insect growth. The tipranavir can inhibit the growth of toxoplasma gondii. Whether HIV inhibition drugs have the effect of inhibiting Toxoplasma gondii or not is clarified, and a reasonable drug treatment scheme can be provided for treating AIDS patients infected by Toxoplasma gondii in the future. Test results show that the form and growth of HFF are not influenced when the concentration of the tipranavir is lower than 30 mu M; the tipranavir has obvious inhibiting effect on toxoplasma gondii and IC thereof509.69 plus or minus 0.91; the nelfinavir does not have antagonism on the sulfadiazine or the pyrimethamine, but the nelfinavir and the sulfadiazine can play an adding role, which indicates that the nelfinavir and the sulfadiazine or the pyrimethamine can be combined to treat toxoplasmosis.
Drawings
FIG. 1 is an IC of the proliferation of Toxoplasma gondii by tipranavir provided by the invention50And (6) obtaining the result.
Detailed Description
The invention provides application of a compound tipranavir shown as a formula I in preparation of a medicament for inhibiting the growth of toxoplasma gondii;
the chemical formula of the compound Tipranavir (Tipranavir) is N- [3- [ (1R) -1- [ (6R) -2-hydroxy-4-oxo-6-phenethyl-6-propyl-5H-pyran-3-yl ] propyl ] phenyl ] -5- (trifluoromethyl) pyridine-2-sulfonamide, CAS No. 174484-41-4. The source of the tipranavir is not particularly limited in the invention, and the conventional commercially available tipranavir product well known to those skilled in the art can be adopted.
The invention also provides application of a composition containing the compounds of the tipranavir and the sulfadiazine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of a composition containing the compounds of the nelfinavir and the pyrimethamine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of a composition containing the compounds of tipranavir, sulfadiazine and pyrimethamine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii.
The invention also provides application of the compound tipranavir shown in the formula I in preparation of a medicine for preventing or treating toxoplasma gondii infection diseases.
The invention also provides application of a composition containing the compounds of the tipranavir and the sulfadiazine shown in the formula I in preparing a medicament for preventing or treating toxoplasma infection diseases.
The invention also provides application of a composition containing the compounds of the nelfinavir and the pyrimethamine shown in the formula I in preparing a medicament for preventing or treating toxoplasma gondii infection diseases.
The invention also provides application of a composition containing the compounds of tipranavir, sulfadiazine and pyrimethamine shown in the formula I in preparing a medicament for preventing or treating toxoplasma infection diseases.
The invention also provides a medicament for preventing or treating toxoplasma infection diseases, which comprises tipranavir and one or two of the following compounds: sulfadiazine and pyrimethamine.
The application of the tipranavir in the preparation of the drug for inhibiting the growth of Toxoplasma gondii is further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
1. Parasites and cell culture
Culturing Toxoplasma gondii RH strain tachyzoite in human foreskin fibroblasts, connecting the RH tachyzoite to a 25T or 75T HFF cell bottle, scraping HFF from the bottom of the bottle by using a cell scraper when about 75% of polypide escapes, transferring cell suspension into a 15mL sterile centrifuge tube, crushing 3-5 times by using an 18G needle, then crushing 3-5 times by using a 27G needle, leading host HFF cells to be crushed under the mechanical action, allowing polypide inside the host cells to escape, filtering the polypide by using a 3 mu m filter, removing cell fragments and counting the Toxoplasma gondii through a blood count plate.
2. Drugs and chemicals
Antiretroviral compounds are purchased from Med Chem Express (Monmouth Junction, NJ, USA). Pyrimethamine and sulfadiazine were purchased from Sigma Chemical Co, (st. louis, MO.) all compounds were dissolved in 100% dimethyl sulfoxide (DMSO) at a final concentration of 10 mM. The final concentration of DMSO, which had no effect on cell viability, did not exceed 0.3% (V/V). Pyrimethamine and sulfadiazine were also dissolved in DMSO as positive controls and DMSO alone as negative controls, respectively.
3. Cell viability assay
Using CellTiter
The AQueous One Solution Cell Proliferation Assay kit observes the toxicity of the HIV inhibitory drug tipranavir on HFF cells. Inoculating HFF cells into a 96-well plate, allowing the HFF cells to culture adherently for 4H, adding corresponding concentrations of tipranavir, continuing to culture for 48H, and adding a tetrazole component [3- (4, 5-dimethylthiozolin-2-yl) -5- (3-carboxymethythiophenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)]And electron coupling reagent (PES), and after incubation at 37 ℃ for 3 hours, absorbance at 490nm was measured with a microplate reader. IC calculation of toxicity of tipranavir to HFF by graphpad prism5 software50(half maximal inhibitory concentration)
HIV inhibiting medicine for resisting toxoplasma proliferation in vitro
The experiment is divided into: experimental group, negative control group, positive control group and blank group. Wherein a negative control group is added with a corresponding volume of DMSO; the positive control component comprises a pyrimethamine positive control group and a sulfadiazine positive control group; the blank was added with the corresponding volume of DMEM solution.
The HFFs grown in 24-well plates were inserted into 5X 10 wells4The freshly released tachyzoites are incubated at 37 ℃ in 5% CO2Culturing under the condition, and after 4h, sucking out the old culture solution and washing away the worm bodies which do not enter the cells. Related concentrations of tipranavir (1. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M) were added to the experimental groups.
Interaction between HIV inhibiting drug-like objects and sulfadiazine or pyrimethamine
Observation of tiranaWhether or not there is some synergy or antagonism between the vir and pyrimethamine or sulfadiazine. Joining HFFs in a 24-well plate to 5X 104The freshly released tachyzoites are incubated at 37 ℃ in 5% CO2Culturing under the condition, discarding the old culture solution after 4h, washing away the insect body which does not enter the cell, and adding corresponding tipranavir and pyrimethamine or sulfadiazine according to the table 1. The toxoplasma growth was then observed.
Table 1 explores whether tipranavir interacts with pyrimethamine or sulfadiazine
After culturing for 5 days in an incubator, collecting infected cells and extracting DNA, and the specific steps are as follows:
(1) the cells cultured adherent should be treated to cell suspension first, then centrifuged at 11,200 Xg for 1min, the supernatant poured off, 200. mu.l of buffer GA added, and shaken to thoroughly suspend
(2) Adding 20 μ l of protease K solution, and mixing
(3) Adding 200 μ l buffer GB, mixing thoroughly, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover.
(4) Add 200. mu.l of absolute ethanol, mix well for 15sec with shaking, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.
(5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging for 30sec at 13,400 Xg, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe.
(6) Adding 500 μ l buffer GD (check whether absolute ethanol has been added before use) into adsorption column CB3, centrifuging for 30sec at 13,400 Xg, pouring off waste liquid, placing adsorption column CB3 into collection tube
(7) To the adsorption column CB3, 600. mu.l of a rinsing liquid PW (previously used, whether or not absolute ethanol was added) was added, and the mixture was centrifuged at 13,400 Xg for 30sec, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube.
(8) Repeat operation step 7
(9) The adsorption column CB3 was returned to the collection tube, centrifuged at 13,400 Xg for 2min, and the waste liquid was discarded. Placing the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material
(10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μ l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 13,400 Xg for 2min, and collecting the solution into the centrifuge tube.
And (3) carrying out worm body quantification by using RT-PCR, wherein the target gene is Toxoplasma gondii B1 gene: the sequence is as follows:
B1-QPCR-F:GGAGGACTGGCAACCTGGTGTCG(SEQ ID NO.1)；
B1-QPCR-R：TTGTTTCACCCGGACCGTTTAGCAG(SEQ ID NO.2)。
application of Applied Blosys 7500Fast Real-Time PCR System and StepOneNuplusTMThe Real-Time PCR System operates.
1. The PCR reaction solution components were prepared as shown in Table 2 (the reaction solution was prepared on ice)
TABLE 2 composition of PCR reaction solution
2. A Real Time PCR reaction was performed. The reaction procedure was as follows: pre-denaturation at 1, 95 ℃ for 30 seconds; 2, 95 ℃ denaturation for 5 seconds; 60 degrees extension for 30 seconds; step 2-3 repeats for 40 cycles; dissolution curve: 95 ℃ for 15 seconds; 60 ℃ for 1 minute; 95 degrees 15 seconds.
Then use 105，104，103，102，101DNA extracted from the worms was used as a standard curve to locate the number of worms, with at least three biological replicates per group.
By graphpadprism 5 software meterIC of computationally related tipranavir on toxoplasma proliferation50The test results are shown in fig. 1 and table 3.
Table 3 percentage growth results for different concentrations of tipranavir and placebo
1. The nelfinavir pair concentration is lower than 30 mu M, and the shape and growth of the HFF are not influenced.
2. The tipranavir has obvious inhibiting effect on toxoplasma gondii and IC thereof50The inhibition was 9.69. + -. 0.91, and the specific inhibition is shown in FIG. 1.
3. The tipranavir does not have antagonism on sulfadiazine or pyrimethamine, but can play an adding role. The specific effects are shown in Table 4.
TABLE 4 inhibition of Toxoplasma by tipranavir in combination with sulfadiazine or pyrimethamine
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (2)
1. The application of a composition containing the compound of the nelfinavir and the sulfadiazine shown in the formula I, a composition containing the compound of the nelfinavir and the pyrimethamine shown in the formula I or a composition containing the compound of the nelfinavir, the sulfadiazine and the pyrimethamine shown in the formula I in preparing a medicament for inhibiting the growth of toxoplasma gondii;
2. a medicament for preventing or treating a toxoplasma infection disease, which comprises the composition of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910743468.2A CN110302199B (en) | 2019-08-13 | 2019-08-13 | Application of tipranavir in preparation of medicine for inhibiting growth of toxoplasma gondii |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910743468.2A CN110302199B (en) | 2019-08-13 | 2019-08-13 | Application of tipranavir in preparation of medicine for inhibiting growth of toxoplasma gondii |
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