CN108434461B - Beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and preparation method thereof - Google Patents
Beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- 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
- A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
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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
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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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
- A61K31/4965—Non-condensed pyrazines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
Abstract
The invention relates to a beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and a preparation method thereof, wherein pyrazinamide and isoniazid rifamycin hydrazone are respectively or jointly included in beta-cyclodextrin to obtain a single inclusion compound or a composition of the inclusion compounds, the bacteriostatic effect of the inclusion compounds on mycobacterium tuberculosis is better than that of a mixture of rifamycin, isoniazid and pyrazinamide, after the inclusion compounds are delivered to a drug administration part in a specific drug administration mode, the inclusion compounds are dispersed, drug molecules respectively act on respective target points to play a role, the first pass effect in the drug taking process is effectively reduced, the drug effect is ensured, and the dosage of the drug is reduced.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and a preparation method thereof.
Background
Tuberculosis (TB) is a chronic infectious disease caused primarily by mycobacterium tuberculosis, transmitted through the respiratory tract. Tubercle bacillus, which primarily infects the lungs, is called tuberculosis (PTB), which is prevalent in numerous countries and regions of the world. In 2015 WHO reported 104 in tuberculosis live reports indicated: tuberculosis is just the largest killer worldwide after aids virus/aids caused by a single infectious pathogen, the majority of young people with the strongest productivity affected by tuberculosis. At present, 4 hundred million people in China are infected with tubercle bacillus once, and the total number of tuberculosis patients is ranked second in the world.
Pyrazinamide (PZA), also known as carbamoylpiperazine, has a molecular formula of C5H5N3O, a relative molecular mass of 123.11, a melting point of 188-192 ℃, and is usually white powder, and has low solubility in water at normal temperature and good solubility in organic solvents of chloroform and dichloromethane. The structural formula of pyrazinamide is shown as the following formula:
pyrazinamide is an important first-line oral antitubercular drug in the treatment of tuberculosis, which was first discovered in 1936 and in 1952 was found to have antibacterial activity. The pyrazinamide is widely used clinically, is used as the most effective antibacterial drug in tuberculosis short-range chemotherapy together with rifampicin and isoniazid, and can shorten the tuberculosis treatment course from 9-12 months to 6 months. In addition, the pyrazinamide has good anticancer property, can effectively inhibit division of tumor cells, reduce the number of the cancer cells and make the tumor cells disappear from ascites. However, direct administration of pyrazinamide has a high incidence of adverse reactions, which easily cause liver damage, and thus liver function needs to be periodically checked during administration.
Through the understanding of the drugs for treating tuberculosis, rifampicin and isoniazid are found to be commonly used as compound-isoprotundine tablets. In the process of compound preparation, a small amount of heterozygote, isoniazid rifamycin hydrazone (English name HYD), is generated, and the structural formula of the heterozygote is shown as the following formula:
isoniazid rifamycin hydrazone appears as one of the impurities generated during the preparation of compound isoprotundine tablets in the 'Chinese pharmacopoeia' of 2015 edition. After the compound is taken by a human body, the HYD residue is detected in gastric juice, excrement and urine, and the substance is not detected in blood. Through the early knowledge of the hybrid in the laboratory and the experience summarized in the synthesis process of other hybrid antibiotics, we suspect that HYD has a certain inhibiting effect on tubercle bacillus, however, no relevant literature describes the synthesis method and physicochemical properties of the hybrid. Therefore, the HYD is synthesized and the biological activity of the HYD is detected, and the result shows that the sterilization effect of the isoniazid rifamycin hydrazone is equivalent to that of rifampicin, so that the research and development of the anti-tuberculosis medicine have important significance and economic value.
Considering that HYD has physicochemical properties similar to rifampicin and poor water solubility, cyclodextrin can be used to improve its solubility after inclusion. Related documents report that after rifampicin is included by beta-cyclodextrin, the solubility of rifampicin can be changed, the absorption of rifampicin can be promoted, and the bioavailability of rifampicin can be improved. In addition, according to the cocktail therapy, several different single-target drugs are used in combination or selectively, and the multi-target drug acting on multiple molecular targets has better curative effect when used for treating complex diseases. Based on the inclusion method in pharmaceutics, drug molecules with different targets are included to form a multi-target drug, because two drug molecules lack commonality, all or part of structural characteristics of the original molecules can be respectively kept in vivo, and then the drug molecules reach a designated administration part through a specific administration mode, the inclusion compound is dispersed, and two or more drug molecules respectively act on respective targets to play roles. Based on the principle, the applicant discloses a thymopentin and rifamycin-isoniazid chitosan clathrate compound earlier, which is specifically disclosed in chinese patent CN 107261106A.
On the basis, the applicant utilizes beta-cyclodextrin to perform inclusion on pyrazinamide and isoniazid rifamycin hydrazone to prepare a single inclusion compound or a composition consisting of a plurality of inclusion compounds, and activity test results show that the inclusion compounds have good antibacterial activity and are generally better than the antibacterial activity of a single component.
Disclosure of Invention
The application aims to provide a beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and a preparation method thereof, and the beta-cyclodextrin inclusion compound is used for including the anti-tuberculosis drugs pyrazinamide, the self-made isoniazid rifamycin hydrazone and the beta-cyclodextrin which are widely used clinically at present, so that the pharmacokinetic property of a small molecular drug is improved, the requirement of directly delivering the drug to a lesion part of a lung is met, and the aims of reducing toxic and side reactions of the drug, improving the bioavailability of the drug, reducing the drug resistance and meeting the clinical drug use requirement are fulfilled. In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone is prepared from one or more of pyrazinamide-beta-cyclodextrin inclusion compound, isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound and pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound through mixing in any ratio.
Furthermore, the molar ratio of pyrazinamide to beta-cyclodextrin in the pyrazinamide-beta-cyclodextrin inclusion compound is 1-3:1, the molar ratio of isoniazid rifamycin hydrazone to beta-cyclodextrin in the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound is 1-3:1, and the molar ratio of pyrazinamide, isoniazid rifamycin hydrazone and beta-cyclodextrin in the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound is 1-3:1-3: 1.
Furthermore, the pyrazinamide-beta-cyclodextrin inclusion compound, the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound and the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound are all nanoparticles.
The preparation method of the beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone comprises the following specific processes:
(a) preparing a pyrazinamide-beta-cyclodextrin inclusion compound by adopting a saturated aqueous solution method or a freeze drying method;
or (b) preparing the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound by adopting a saturated aqueous solution method or a dry grinding method;
or (c) uniformly mixing the alkali solution of isoniazid rifamycin hydrazone with the alkali solution of pyrazinamide, adding the aqueous solution of sodium tripolyphosphate, slowly dripping the obtained mixed solution into the saturated beta-cyclodextrin acetic acid solution, fully stirring, standing and refrigerating at-5 to 4 ℃ for 24 to 48 hours to obtain the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound;
or (d) uniformly mixing at least two of the pyrazinamide-beta-cyclodextrin inclusion compound, the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound and the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound according to any ratio to obtain the inclusion compound composition.
Further, the saturated aqueous solution method specifically comprises the following steps: firstly, preparing saturated aqueous solution of beta-cyclodextrin, then dissolving a medicament (pyrazinamide or isoniazid rifamycin hydrazone and the like) in ethanol with the mass of 8-15 times of that of the medicament, then mixing and stirring the two solutions uniformly, placing the mixed solution at 0-4 ℃, standing and refrigerating for 18-48h, and finally sequentially filtering, washing and drying at 35-55 ℃.
Further, the freeze drying method specifically comprises the following steps: firstly, dissolving beta-cyclodextrin in hot water with the mass of 50 times and the temperature of 70-100 ℃, then adding a medicament (pyrazinamide and the like) into the beta-cyclodextrin, fully stirring the mixture, then freezing the obtained mixed solution at the low temperature of-5 ℃ for 8-15h, finally taking out the mixed solution, washing the medicine which is not encapsulated by dichloromethane, and removing the dichloromethane.
Further, the dry grinding method specifically comprises the following steps: mixing beta-cyclodextrin and medicine (isoniazid rifamycin hydrazone, etc.), grinding, and drying at 35-60 deg.C for 8-12 hr.
Further, in the step (c), the obtained mixed solution is slowly dripped into a saturated beta-cyclodextrin acetic acid solution, and the concentration of the beta-cyclodextrin in the formed mixed solution is 1.5-6.0 g/L. The experimental result shows that the beta-cyclodextrin nano-particles can be formed only when the concentration is within a specific concentration range, namely the concentration range of the beta-cyclodextrin in the reaction liquid is 1.5-6.0 g/L.
Further, the alkali used for preparing the alkali solution of isoniazid rifamycin hydrazone and the alkali solution of pyrazinamide in the step (c) is selected from NaOH, KOH and Na2CO3、K2CO3And sodium tert-butoxide and potassium tert-butoxide.
Further, the concentration of the alkali solution of isoniazid rifamycin hydrazone in the step (c) is 0.001-0.004g/mL, the concentration of the alkali solution of pyrazinamide is 0.01-0.02g/mL, and the concentration of the aqueous solution of sodium tripolyphosphate is 0.001-0.004 g/mL.
Compared with the prior art, the invention has the following unexpected beneficial effects:
(1) the preparation method of the pyrazinamide and isoniazid rifamycin hydrazone clathrate compound is developed, and isoniazid rifamycin hydrazone which is originally used as an impurity in the preparation process of the compound tablet is developed into a novel anti-tuberculosis clathrate compound, and a better effect is achieved;
(2) the drugs (pyrazinamide and isoniazid rifamycin hydrazone) with different action targets are included together through simple inclusion, so that the beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone and the composition thereof are obtained, and experiments show that the antibacterial effect of the beta-cyclodextrin inclusion compound on mycobacterium tuberculosis is better than that of traditional drugs such as pyrazinamide, rifamycin and isoniazid or the mixture thereof;
(3) after the prepared inclusion compound is delivered to a drug administration part in a specific drug administration mode, the inclusion compound disperses two or more drug molecules to respectively act on respective target points to play a role, thereby effectively avoiding the first pass effect of the drug metabolism in vivo, and reducing the dosage of the drug while ensuring the drug effect;
(4) compared with a single inclusion compound, the composite inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone has weakened drug resistance and better effect.
Drawings
Fig. 1 is a DSC diagram of pyrazinamide (a), β -cyclodextrin (B), a physical mixture of pyrazinamide and β -cyclodextrin (C), and pyrazinamide- β -cyclodextrin inclusion compound (D) prepared in example 1.
Detailed Description
In order to make those skilled in the art fully understand the technical solutions and advantages of the present invention, the following embodiments are further described.
The invention calculates the yield of the inclusion compound and the inclusion rate as follows (three times of parallel measurement and average value):
wherein, the object is pyrazinamide, isoniazid rifamycin hydrazone, pyrazinamide + isoniazid rifamycin hydrazone and the like, and the subject is beta-cyclodextrin.
Example 1
The pyrazinamide-beta-cyclodextrin inclusion compound is prepared by respectively adopting a saturated aqueous solution method and a freeze drying method, and the specific process is as follows:
(1) saturated aqueous solution process
1g of beta-cyclodextrin is added into a beaker, and then a proper amount of water is added to be stirred into a saturated solution. Pyrazinamide (about 0.108g) having the same molar number as that of β -cyclodextrin was precisely weighed, and added to 3mL of ethanol, and slightly heated to dissolve it, thereby obtaining an ethanol solution of pyrazinamide. Under the condition of stirring, dropwise adding an ethanol solution of pyrazinamide into the saturated solution of the beta-cyclodextrin, stirring for 0.5-1h, placing in an environment of 0-4 ℃ for refrigeration and standing for 18-24h, filtering, washing with a small amount of distilled water, and then transferring into an oven of 35-55 ℃ for drying to obtain the inclusion compound. The obtained inclusion compound is stored at low temperature in a closed and dry place.
The inclusion rate and the yield of the clathrate were measured by the above-described methods, and were 84.5% and 82.13%, respectively.
(2) Freeze drying method
Weighing 1g of beta-cyclodextrin, dissolving the beta-cyclodextrin in hot water at 70-100 ℃, adding pyrazinamide (about 0.108g) with the same mole number as that of the beta-cyclodextrin, stirring for 0.5-1h, placing the mixed solution in a refrigerator at-5-0 ℃ for overnight freeze drying, washing out unencapsulated pyrazinamide with dichloromethane, and finally removing residual solvent to obtain white inclusion compound powder. The obtained inclusion compound is stored at low temperature in a closed and dry place.
The inclusion rate and the yield of the clathrate were determined according to the above methods, and were 81.33% and 85.37%, respectively.
Example 2
The isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound is prepared by respectively adopting a saturated aqueous solution method and a dry grinding method, and the specific process is as follows:
(1) saturated aqueous solution process
0.1g of beta-cyclodextrin is added into a beaker, and then a proper amount of water is added to be stirred into a saturated solution. Accurately weighing isoniazid rifamycin hydrazone (about 0.074g) with the mole number equal to that of the beta-cyclodextrin, dissolving the isoniazid rifamycin hydrazone in a small amount of ethanol, adding the ethanol solution of the isoniazid rifamycin hydrazone into the saturated solution of the beta-cyclodextrin, stirring for 0.5h, placing the mixture in an environment with the temperature of 0-4 ℃ for refrigeration and standing for 24h, filtering, washing with a small amount of ethanol, and then transferring the mixture into a drying oven with the temperature of 40 ℃ for drying to obtain the inclusion compound. The obtained inclusion compound is stored at low temperature in a closed and dry place.
The inclusion rate and the yield of the inclusion compound were measured by the above methods, and as a result, they were 81.47% and 81.107%, respectively.
(2) Dry milling process
Weighing 0.1g of beta-cyclodextrin, putting the beta-cyclodextrin into a mortar, adding isoniazid rifamycin hydrazone (about 0.074g) with the mole number equal to that of the beta-cyclodextrin, grinding while adding, continuing to grind for 30-60min until the mixture is fully ground, and then transferring the mixture into an oven at 35-55 ℃ to dry to obtain the inclusion compound. The obtained inclusion compound is stored at low temperature in a closed and dry place.
The inclusion rate and the yield of the clathrate were determined according to the above methods, and were 71.95% and 87.03%, respectively.
Example 3
The method for preparing the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound comprises the following steps:
a. dissolving 10mg of beta-cyclodextrin in 5mL of acetic acid solution with volume fraction of 0.25%, and fully swelling for 24 hours until the beta-cyclodextrin is completely dissolved to obtain a beta-cyclodextrin solution;
b. dissolving 1.7mg of pyrazinamide in 0.1mL of NaOH solution with the concentration of 0.1mol/L, and fully dissolving to obtain pyrazinamide solution;
c. dissolving 3mg of isoniazid rifamycin hydrazone in 1mL of NaOH solution with the concentration of 0.1mol/L, taking out 0.3mL of the solution, adding the solution into the pyrazinamide solution prepared in the step b, adding 1mL of sodium Tripolyphosphate (TPP) aqueous solution with the concentration of 2g/L, and uniformly stirring to obtain a mixed solution;
d. and (c) slowly dripping the mixed solution dissolved with pyrazinamide, isoniazid rifamycin hydrazone and TPP prepared in the step (c) into the swelled beta-cyclodextrin solution obtained in the step (a), stirring at room temperature for 30min, adding a small amount of lactose as a protective agent, and freeze-drying at-5 to 0 ℃ for 36 to 48h to obtain the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound. The obtained inclusion compound is stored at low temperature in a closed and dry place.
The inclusion rate and the yield of the clathrate were determined by the above-described methods, and were 79.85% and 82.235%, respectively.
Example 4
The pyrazinamide- β -cyclodextrin inclusion compound prepared in example 1 and the isoniazid rifamycin hydrazone- β -cyclodextrin inclusion compound prepared in example 2 were mixed by mass to obtain an inclusion compound composition.
DSC analysis test
DSC analysis was performed on pyrazinamide (a), β -cyclodextrin (B), a physical mixture of pyrazinamide and β -cyclodextrin (molar ratio of pyrazinamide to β -cyclodextrin is 1:1, C), and the pyrazinamide- β -cyclodextrin inclusion compound (D) prepared in example 1, respectively, and the analysis procedures were as follows:
precisely weighing about 4-5mg of each dried sample, uniformly spreading the sample in an aluminum foil box special for DSC test, sealing by a pressing cover of a tablet press, and testing by using a blank aluminum foil box as a control, wherein the scanning speed is 20 ℃/min and the range is 25-240 ℃. The obtained DSC thermogram is shown in figure 1, wherein A is pyrazinamide, B is beta-cyclodextrin, C is a physical mixture of the pyrazinamide and the beta-cyclodextrin, and D is pyrazinamide-beta-cyclodextrin inclusion compound.
As can be seen from FIG. 1, pyrazinamide has an endothermic peak at a melting point of 190 ℃ which is a melting point peak; beta-cyclodextrin exhibits an endothermic peak at a temperature of about 105 ℃; the physical mixture of the two has 2 endothermic peaks at 105 ℃ and 190 ℃, which indicates that the physical mixture is simply mixed without forming new substances. By reversing the inclusion complex prepared in example 1, the melting point peak of pyrazinamide in the DSC curve disappeared, indicating that pyrazinamide- β -cyclodextrin inclusion complex did form. Similar tests were also performed on the inclusion compounds prepared in examples 2 to 3, leading to the same conclusion.
Drug resistance testing
The isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound prepared in example 2 was used as a sample, and the lyophilized product was diluted to different multiples, and the drug resistance to the cultured mycobacterium tuberculosis strain was tested separately and compared with a physical mixture of rifamycin and isoniazid (the mass ratio of rifamycin to isoniazid was 1: 1).
The experimental method comprises the following steps: the clathrate and the mixture are respectively prepared into liquid medicine with the concentration of 1mg/mL, and the liquid medicine is sealed by a sterile film for standby after being sterilized.
(1) 100 μ L of 7H9 medium was added to each well of a sterile 96-well plate (1-12 wells). Add 100. mu.L of stock solution of antituberculotic diluted appropriately into well 1, and dilute to well 12 in duplicate. The final concentration of the medicines in each hole is 17-0.017 mu g/mL of the mixture of rifamycin and isoniazid, and 17-0.017 mu g/mL of isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound.
(2) 2 drops of 10% (mass fraction) Tween-80 saline and clinically isolated fresh culture of 2-3 weeks old were added to the bottom of a glass sterilizer, ground to cream-like, diluted with saline to turbidity (1mg/mL) in a No. 1 Mach turbidimeter, and 100. mu.L of the culture was inoculated into 1-8 wells after 1: 20-fold dilution in 7H9 medium. Blank wells are media controls.
(3) Sealing the plate with sterile membrane, placing in a wet box, culturing at 37 deg.C for 5d, adding filter sterilized and 0.1g/L resazurin color developing solution 30 μ L to the 12 th well on day 6, and incubating for 24 h. If the blank hole (without medicine) becomes pink, the same amount of resazurin developing solution is added to other holes, and the color change is recorded after 24 h. If the blank well is still blue, it is observed on days 7 and 9, respectively. The color change from blue to pink is indicative of bacterial growth.
The isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound prepared in example 2 was diluted to different concentrations and tested for its resistance to mycobacterium tuberculosis, and compared with a mixture of rifamycin and isoniazid at the same concentrations, and the test results are shown in table 1.
TABLE 1 comparison of drug resistance of Inclusion compounds and mixtures of isoniazid rifamycin hydrazone and beta-cyclodextrin
Remarking: r is resistance to Mycobacterium tuberculosis, i.e., no activity; s is sensitivity to M.tuberculosis, i.e.activity (concentration unit is mug/mL).
As can be seen from Table 1, the bacteriostatic effect begins to appear when the concentration of the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound is 0.133 mug/mL, and the bacteriostatic effect appears when the concentration of the mixture of rifamycin and isoniazid is 1.063 mug/mL, so that the isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound is considered to have better bacteriostatic effect than the mixture of rifamycin and isoniazid.
Experiment of antibacterial Activity
Levofloxacin-chitosan inclusion compound, pyrazinamide-beta-cyclodextrin inclusion compound, isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound and pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound with the same inclusion ratio are respectively selected for carrying out antibacterial activity comparison tests. The 7H9 medium for the antituberculotic was provided by infectious disease Hospital, Wuhan City. 7H9 culture medium without antituberculosis drugs is used as blank control, the experimental method is micropore chromogenic method, and blind method arrangement test is adopted. The culture medium without antituberculosis drugs is used as a control group, the HR37V standard strain is used as a specimen, the color development condition of the culture medium is observed along with the time, the color development condition of the culture medium reflects the growth conditions of different kinds of tubercle bacillus, and the bacteriostatic activity of the drugs on the tubercle bacillus is further illustrated. The lowest concentration of the medium that can exhibit a blue color is the MIC value of the drug. The smaller the MIC value is, the stronger the drug effect is; the higher the MIC value, the worse the drug effect. The results of the antimicrobial activity test are shown in table 2 below:
TABLE 2 results of antibacterial Activity of different Inclusions
As can be seen from Table 2, all four clathrates have good bactericidal activity, consistent with our expectation. Compared with the existing levofloxacin-chitosan inclusion compound, the three newly prepared inclusion compounds have certain advantages; pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin has the relevant characteristics of pyrazinamide-beta-cyclodextrin and isoniazid rifamycin hydrazone-beta-cyclodextrin.
Claims (5)
1. A beta-cyclodextrin inclusion compound of pyrazinamide and isoniazid rifamycin hydrazone is characterized in that: the inclusion compound is a pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound, wherein the molar ratio of pyrazinamide, isoniazid rifamycin hydrazone and beta-cyclodextrin is 1-3:1-3:1, and the inclusion compound is nanoparticles.
2. The process for preparing β -cyclodextrin inclusion complex of pyrazinamide, isoniazid rifamycin hydrazone as claimed in claim 1, comprising the steps of: uniformly mixing the alkali solution of isoniazid rifamycin hydrazone with the alkali solution of pyrazinamide, adding the aqueous solution of sodium tripolyphosphate, slowly dripping the obtained mixed solution into the saturated beta-cyclodextrin acetic acid solution, fully stirring, standing at-5 to 4 ℃, and refrigerating for 24 to 48 hours to obtain the pyrazinamide-isoniazid rifamycin hydrazone-beta-cyclodextrin inclusion compound.
3. The method of claim 2, wherein: slowly and dropwise adding the obtained mixed solution into a saturated beta-cyclodextrin acetic acid solution to form a mixed solution, wherein the concentration of the beta-cyclodextrin in the formed mixed solution is 1.5-6.0 g/L.
4. The method of claim 2, wherein: the alkali used for preparing the alkali solution of isoniazid rifamycin hydrazone and the alkali solution of pyrazinamide is selected from NaOH, KOH and Na2CO3、K2CO3And sodium tert-butoxide and potassium tert-butoxide.
5. The method of claim 2, wherein: the concentration of the alkali solution of isoniazid rifamycin hydrazone is 0.001-0.004g/mL, the concentration of the alkali solution of pyrazinamide is 0.01-0.02g/mL, and the concentration of the aqueous solution of sodium tripolyphosphate is 0.001-0.004 g/mL.
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| CN107216343A (en) * | 2017-05-26 | 2017-09-29 | 武汉工程大学 | A kind of rifamycin isoniazid heterozygosis medicine and preparation method thereof |
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Non-Patent Citations (3)
| Title |
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| "Inclusion complexes of β-cyclodextrin with pyrazinamide and piperazine: Crystallographic and theoretical studies";Thammarat Aree et al.;《Supramolecular Chemistry》;20090630;第21卷(第5期);摘要，图1 * |
| Dan He et al.,."Molecular encapsulation of rifampicin as an inclusion complex of hydroxypropyl-β-cyclodextrin: Design;characterization and in vitro dissolution".《Colloids and Surfaces B: Biointerfaces》.2013,第103卷580–585. * |
| Thammarat Aree et al.."Inclusion complexes of β-cyclodextrin with pyrazinamide and piperazine: Crystallographic and theoretical studies".《Supramolecular Chemistry》.2009,第21卷(第5期),384–393. * |
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