CN117180303A - Application of full-guanidino-substituted cyclodextrin as antibiotic adjuvant - Google Patents
Application of full-guanidino-substituted cyclodextrin as antibiotic adjuvant Download PDFInfo
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- 229920000858 Cyclodextrin Polymers 0.000 title claims abstract description 167
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical class O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 239000003474 antibiotic adjuvant Substances 0.000 title claims abstract description 28
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 69
- 229960004853 betadex Drugs 0.000 claims abstract description 69
- 239000003814 drug Substances 0.000 claims abstract description 58
- 229940079593 drug Drugs 0.000 claims abstract description 56
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 24
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 24
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 13
- 229940123208 Biguanide Drugs 0.000 claims abstract description 4
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 42
- 229960001225 rifampicin Drugs 0.000 claims description 42
- 230000003115 biocidal effect Effects 0.000 claims description 40
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 25
- -1 amino cyclodextrin Chemical compound 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 17
- 241000191967 Staphylococcus aureus Species 0.000 claims description 13
- 241000588626 Acinetobacter baumannii Species 0.000 claims description 11
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims description 11
- 229960003085 meticillin Drugs 0.000 claims description 11
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims description 9
- RBZRMBCLZMEYEH-UHFFFAOYSA-N 1h-pyrazol-1-ium-1-carboximidamide;chloride Chemical compound Cl.NC(=N)N1C=CC=N1 RBZRMBCLZMEYEH-UHFFFAOYSA-N 0.000 claims description 8
- 206010059866 Drug resistance Diseases 0.000 claims description 6
- 150000001540 azides Chemical class 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 6
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- 238000005576 amination reaction Methods 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical group II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 239000011630 iodine Substances 0.000 claims description 3
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 241000589562 Brucella Species 0.000 claims description 2
- 241000194031 Enterococcus faecium Species 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 claims description 2
- 239000004098 Tetracycline Substances 0.000 claims description 2
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- 238000000034 method Methods 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 16
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine group Chemical group NC(=N)N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 12
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- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 8
- 239000000047 product Substances 0.000 description 8
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- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 5
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 4
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- 239000002671 adjuvant Substances 0.000 description 3
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
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- OCVXZQOKBHXGRU-UHFFFAOYSA-N iodine(1+) Chemical compound [I+] OCVXZQOKBHXGRU-UHFFFAOYSA-N 0.000 description 2
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- 229920001450 Alpha-Cyclodextrin Polymers 0.000 description 1
- XNCOSPRUTUOJCJ-UHFFFAOYSA-N Biguanide Chemical compound NC(N)=NC(N)=N XNCOSPRUTUOJCJ-UHFFFAOYSA-N 0.000 description 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 1
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 208000032376 Lung infection Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
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- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 1
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000012984 antibiotic solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
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- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 1
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Abstract
The invention discloses an application of a full-guanidino-substituted cyclodextrin as an antibiotic adjuvant, which is characterized in that the full-guanidino-substituted cyclodextrin is used as the antibiotic adjuvant to reduce the minimum inhibitory concentration of antibiotics on drug-resistant bacteria, the application method is that the full-guanidino-substituted cyclodextrin and the antibiotics are sterilized in a combined way, the full-guanidino-substituted cyclodextrin is full-6-biguanide or full-6-Shan Guaji-substituted cyclodextrin, and the general formula of the full-6-biguanide-beta-cyclodextrin is as follows: (C) 7 H 13 N 5 O 4 ) n N=6, 7,8, the general formula of the all-6-Shan Guaji-beta-cyclodextrin is (C 7 H 13 N 3 O 4 ) n ，n＝6,7,8。
Description
Technical Field
The invention belongs to the field of antibiotic adjuvants, and relates to an application of full guanidine substituted ring paste as an antibiotic adjuvant.
Background
The discovery of antibiotics has resulted in a significant reduction in mortality in patients with bacterial infections. However, abuse of antibiotics has also led to the emergence of multi-drug resistant bacteria, which has led to an increasing impairment of the therapeutic efficacy of antibiotics. In addition, because the development of novel antibiotics is hindered and the current situation that the existing medicines are difficult to cope with drug-resistant bacteria infection, the antibiotic adjuvant capable of restoring the curative effect of the existing antibiotics becomes a new strategy with great potential for coping with the development of bacterial drug resistance. Electropositive cations can affect cell membrane permeability, with the potential to restore antibiotic sensitivity as antibiotic adjuvants.
Cyclodextrin is a cyclic oligosaccharide compound generated by enzymatic degradation of starch, and in the cyclic structure of the cyclodextrin, the hydroxyl group on a glucose unit is oriented to the outer side of the cyclodextrin to form the hydrophilic outer edge of the cyclodextrin; proton hydrogen on methylene and methine points to the inner side to form a hydrophobic inner cavity of cyclodextrin, and the cyclodextrin is a unique amphiphilic structure with hydrophilic outer edge inner cavity. Common cyclodextrins are three types of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin. In aqueous solution, the hydrophobic inner cavity of cyclodextrin can produce enveloping action on nonpolar small molecules with proper size and shape, and cyclodextrin is applied to develop drug delivery systems at the earliest, and can wrap insoluble drugs in a cavity structure to form inclusion compounds so as to improve the solubility and bioavailability of the insoluble drugs. In addition, cyclodextrin inclusion compounds can also improve physical and thermal stability of the drug and help control drug toxicity. Has small irritation to mucous membrane and eyes, and almost no toxicity when taken orally. They can also be used as medicaments for oral, ocular, dermal, nasal and rectal administration, masking the taste of unpleasant medicaments and reducing irritation to the eye, gastrointestinal tract or skin. Cyclodextrin has become one of the most valuable drug delivery vehicles, allowing more and more insoluble drugs to pass clinical trials, and many cyclodextrin-drug inclusion complexes have been marketed. Although cyclodextrins have several advantages, cyclodextrins are uncharged, limiting their use in drug-resistant bacterial killing. Cation modification can introduce positive charge into the cyclodextrin. In addition, guanidine drugs have long been used clinically. Therefore, the guanidyl modification of the cyclodextrin has important practical and clinical significance.
Disclosure of Invention
In view of this, the present invention provides the use of a full guanidino-substituted ring paste as an antibiotic adjuvant. The invention specifically provides the following technical scheme:
the preparation method comprises the steps of using the full-guanidino-substituted cyclodextrin as an antibiotic adjuvant, killing drug-resistant bacteria which have generated drug resistance to antibiotics, reducing the use amount of the antibiotics and the minimum inhibitory concentration of the antibiotics to the drug-resistant bacteria, wherein the full-guanidino-substituted cyclodextrin is full-6-biguanide or full-6-Shan Guaji-substituted cyclodextrin, and the general formula of the full-6-biguanide-beta-cyclodextrin is as follows: (C) 7 H 13 N 5 O 4 ) n N=6, 7,8, the general formula of the all-6-Shan Guaji-beta-cyclodextrin is (C 7 H 13 N 3 O 4 ) n ，n＝6,7,8。
Furthermore, when the whole guanidyl substituted ring paste is used as an antibiotic adjuvant, the mass ratio of the antibiotic to the whole guanidyl substituted ring paste is 1:32-1:128000.
Further, the drug-resistant bacteria are: drug-resistant pseudomonas aeruginosa, drug-resistant staphylococcus aureus, drug-resistant enterococcus faecium, drug-resistant klebsiella pneumoniae, drug-resistant acinetobacter baumannii, drug-resistant escherichia coli, drug-resistant brucella or drug-resistant tubercle bacillus.
Further, the antibiotic is rifampin, tetracycline, streptomycin or doxycycline.
Further, aiming at the multi-drug resistant pseudomonas aeruginosa, the mass ratio of the antibiotic to the full guanidyl substituted ring paste is 1:64-1:8196; the mass ratio of the antibiotic to the full guanidine-substituted ring paste used in a combined way is 1:32-1:4096 for the multi-drug resistant Acinetobacter baumannii, and the mass ratio of the antibiotic to the full guanidine-substituted ring paste used in a combined way is 1:2000-1:128000 for the methicillin-resistant staphylococcus aureus.
Further, aiming at the multi-drug resistant pseudomonas aeruginosa, the mass ratio of the combined use of the antibiotics and the full guanidyl substituted ring paste is 1:64-1:4096, and the mass ratio of the combined use of the antibiotics and the Shan Guaji substituted ring paste is 1:128-1:8196; aiming at the multi-drug resistant Acinetobacter baumannii, the mass ratio of the antibiotic to the full guanidine-substituted ring paste is 1:32-1:4096, and the mass ratio of the antibiotic to the Shan Guaji-substituted ring paste is 1:128-1:2048; the mass ratio of the antibiotic to the full guanidine-substituted ring paste for the methicillin-resistant staphylococcus aureus is 1:2000-1:128000, and the mass ratio of the antibiotic to the full guanidine-substituted ring paste for the methicillin-resistant staphylococcus aureus is 1:4000-1:32000.
Further, the preparation method of the full guanidine group substituted ring paste comprises the following steps:
1) Carrying out amination modification on cyclodextrin to obtain amino cyclodextrin;
2) Amino cyclodextrin reacts with dicyandiamide to obtain the full-6-biguanidino-beta-cyclodextrin or amino cyclodextrin reacts with 1H-pyrazole-1-formamidine hydrochloride to obtain the full-6-Shan Guaji-beta-cyclodextrin.
Further, step 1) is to replace the hydroxyl group at the 6-position of cyclodextrin with iodine, then replace iodine with azide, and finally reduce the azide to amino.
Further, the reaction conditions of step 1) are: the solvent of the reaction system is N, N-dimethylformamide, the reaction temperature is between room temperature and 70 ℃, and the reaction time is between 24 and 72 hours.
Further, the reaction conditions of the amino cyclodextrin and dicyandiamide in step 2) are as follows: the mass ratio of the amino cyclodextrin to the dicyandiamide is 0.3-0.5, the reaction system is aqueous solution, the reaction temperature is 110-130 ℃, and the reaction time is 24-72 h.
Further, the reaction conditions of the amino cyclodextrin and the 1H-pyrazole-1-carboxamidine hydrochloride in the step 2) are as follows: the mass ratio of the amino cyclodextrin to the 1H-pyrazole-1-formamidine hydrochloride is 0.1-0.3, the solvent of the reaction system is N, N-dimethylformamide, the reaction temperature is 60-80 ℃, and the reaction time is 48-96H.
The invention has the beneficial effects that: the method of the invention carries out full guanidine substitution on the cyclodextrin, and the modified cyclodextrin has high biological safety, and the full guanidine substitution cyclodextrin is used as an antibiotic adjuvant, so that the sensitivity of various drug-resistant bacteria to various antibiotics can be recovered, and the new bacterial drug resistance can not be caused. In addition, the cationic cyclodextrin small molecule can be used as an antibiotic adjuvant to cooperate with antibiotics for treating various clinical bacterial infection diseases. The invention provides a preparation method of cyclodextrin with excellent performance, high biocompatibility and controllable operation, which is used as an antibiotic adjuvant for killing drug-resistant bacteria and recovering sensitivity to drug-resistant antibiotics.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention clearer, the present invention provides the following drawings:
FIG. 1 is a diagram of a full-6-biguanidino-beta-cyclodextrin 13 C nuclear magnetic resonance diagram.
FIG. 2 is a diagram of a full-6-Shan Guaji-beta-cyclodextrin 13 C nuclear magnetic resonance diagram.
FIG. 3 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin resistant multidrug resistant Pseudomonas aeruginosa.
FIG. 4 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin resistant multi-drug resistant Acinetobacter baumannii.
FIG. 5 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin against methicillin-resistant Staphylococcus aureus.
FIG. 6 is a graph showing the results of in vivo synergy experiments with holigidic substituted cyclodextrin and rifampicin resistant multidrug resistant P.aeruginosa.
FIG. 7 minimum inhibitory concentration of holigidic substituted cyclodextrin on multidrug resistant P.aeruginosa.
FIG. 8 cytotoxicity test of holguanidyl substituted cyclodextrins.
FIG. 9 shows the route for preparing the adjuvant of the whole guanidyl substituted cyclodextrin micromolecule antibiotics.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
FIG. 9 shows the preparation route of the whole guanidine-substituted cyclodextrin small molecule antibiotic adjuvant. Firstly, substituting hydroxyl on the 6 th position of cyclodextrin with iodine, substituting iodine with azide, reducing the azide into amino to obtain aminated modified cyclodextrin, and reacting with dicyandiamide to obtain biguanidino cyclodextrin; reacting with 1H-pyrazole-1-formamidine hydrochloride to obtain guanidino cyclodextrin. The cation substituted cyclodextrin is used as an antibiotic adjuvant, so that the sensitivity of the drug-resistant bacteria to the drug-resistant antibiotic can be recovered, and new bacterial drug resistance can not be caused.
Example 1
Preparation of all-6-biguanidino-beta-cyclodextrin:
1) Amination modification of cyclodextrin
Iodine (I) 2 ) 221mmol and 175mmol of Triphenylphosphine (TPP) are added into 250mL of anhydrous N, N-Dimethylformamide (DMF) solution of Cyclodextrin (CD) (62 mmol of sugar ring), the mixture is reacted for 48h at 70 ℃ under the protection of nitrogen, 25% sodium methoxide/methanol solution with mass fraction is slowly added under the condition of ice-water bath, the pH is regulated to 9, the mixture is stirred uniformly, a large amount of yellow precipitate is generated at the moment, the mixture is centrifuged, the methanol is washed, and the lower precipitate is collected for vacuum drying, thus obtaining the product CD-I. NaN is processed 3 (92.1 mmol) was added to a solution of CD-I (36.8 mmol of sugar ring) in DMF (200 mL), reacted at 60℃for 24h under nitrogen protection, deionized water was added, centrifuged, washed with water, the lower precipitate was collected, and freeze-dried to give CD-N product 3 . TPP (122 mmol) was added to the product CD-N 3 (53 mmol sugar ring) in DMF (200 mL) under nitrogen protection at room temperature for 48h, precipitating with ethanol, centrifuging, washing with ethanol, collecting the lower precipitate, and vacuum drying to obtain CD-NH product 2 。
2) Amino cyclodextrin biguanide
Firstly, carrying out hydrochloride treatment on amino cyclodextrin, dissolving 1g of all-6-amino-beta-cyclodextrin in 40mL (0.3 mol/L) hydrochloric acid, stirring at room temperature until the all-6-amino-beta-cyclodextrin is completely dissolved, and precipitating with ethanol to obtain all-6-amino-beta-cyclodextrin hydrochloride (CD-NH) 2 ·HCl)。
Dissolving 1g of the full-6-amino-beta-cyclodextrin hydrochloride obtained in the step 2) in 15mL of water, adding 2.1g of Dicyandiamide (DICY), reacting for two days at 120 ℃, adding water, dialyzing, and freeze-drying to obtain the full-6-biguanidino-beta-cyclodextrin.
FIG. 1 is a diagram of a full-6-biguanidino-beta-cyclodextrin 13 C nuclear magnetic resonance diagram. 13 C NMR(DMSO-d6):40.298(C 6f )；68.205(C 2b )；71.752(C 3c )；72.300(C 5e )；82.551(C 4d )；101.728(C 1a )；160.013(C 7g ). The characteristic peak accords with the structural formula of the full-6-biguanide-beta-cyclodextrin.
Example 2
Preparation of all-6-Shan Guaji-beta-cyclodextrin:
1) Amination modification of cyclodextrin
Iodine (I) 2 ) 221mmol and 175mmol of Triphenylphosphine (TPP) are added into 250mL of anhydrous N, N-Dimethylformamide (DMF) solution of Cyclodextrin (CD) (62 mmol of sugar ring), the mixture is reacted for 48h at 70 ℃ under the protection of nitrogen, 25% sodium methoxide/methanol solution with mass fraction is slowly added under the condition of ice-water bath, the pH is regulated to 9, the mixture is stirred uniformly, a large amount of yellow precipitate is generated at the moment, the mixture is centrifuged, the methanol is washed, and the lower precipitate is collected for vacuum drying, thus obtaining the product CD-I. NaN is processed 3 (92.1 mmol) was added to a solution of CD-I (36.8 mmol of sugar ring) in DMF (200 mL), reacted at 60℃for 24h under nitrogen protection, deionized water was added, centrifuged, washed with water, the lower precipitate was collected, and freeze-dried to give CD-N product 3 . TPP (122 mmol) was added to the product CD-N 3 (53 mmol sugar ring) in DMF (200 mL) under nitrogen protection at room temperature for 48h, precipitating with ethanol, centrifuging, washing with ethanol, collecting the lower precipitate, and vacuum drying to obtain CD-NH product 2 。
2) Amino cyclodextrin monoguanidine
1g of all-6-amino-. Beta. -cyclodextrin was dissolved in 50mL of anhydrous DMF, 1H-pyrazole-1-carboxamidine hydrochloride (6.15 g) was added and reacted at 70℃for three days, and N, N-diisopropylethylamine (8.25 mL) was added in three portions over three days. After the reaction, isopropyl ether is used for 2 hours, the supernatant is poured out, deionized water and ethanol are used for washing the precipitate, the precipitate is centrifuged, and vacuum drying is carried out on the precipitate, so that the full-6-Shan Guaji-beta-cyclodextrin is obtained.
FIG. 2 is a diagram of all-6-Shan Guaji-beta-cyclodextrin 13 C nuclear magnetic resonance diagram. 13 CNMR(DMSO-d6)：42.275(C 6f )；70.883(C 2b )；71.755(C 3c )；72.462(C 5e )；82.607(C 4d )；101.819(C 1a )；157.753(C 7g ). The characteristic peak accords with the molecular structural formula of the full-6-Shan Guaji-beta-cyclodextrin.
Example 3
Synergistic antimicrobial test of hologuanyl substituted ring pastes with antibiotics:
all-6-biguanidino-beta-cyclodextrin and all-6-Shan Guaji-beta-cyclodextrin are collectively referred to as all-guanidino-substituted cyclodextrins. The synergistic antibacterial effect of different antibiotics and the holo-guanidino-substituted cyclodextrin is evaluated by a chessboard dilution method, and the method is as follows:
firstly, different concentrations of rifampicin solution, full-6-biguanidino-beta-cyclodextrin solution and full-6-Shan Guaji-beta-cyclodextrin solution are obtained through continuous half-dilution for standby.
The whole guanidino-substituted cyclodextrin solution was then added in 96-well plates with decreasing concentrations in the x-axis direction. The same volume of antibiotic solution was added with decreasing concentration in the y-axis direction in order to give a bacterial concentration of 1X 10 per well 5 CFU/mL. The 96-well plate was then placed in a 37℃incubator for 12 hours, and absorbance at 600nm was measured using a microplate reader.
The interaction between the antibiotic and the holo-guanidino-substituted cyclodextrin was evaluated by a Fractional Inhibitory Concentration Index (FICI) and calculated as formula (1): fici=fic A +FIC B Wherein the FIC A MIC of whole guanidino substituted cyclodextrin/MIC of antibiotic alone, FIC when combined B MIC of antibiotic at combination/MIC of antibiotic alone, when fici.ltoreq.0.5 is defined as synergy between a and B, 0.5<FICI.ltoreq.1 is defined as the additive effect between A and B; when FICI>1 is defined as no relationship between the two.
FIG. 3 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin resistant multidrug resistant Pseudomonas aeruginosa.
As can be seen from FIG. 3 (left), when all-6-biguanide-beta-cyclodextrin and rifampicin act together on multi-drug resistant Pseudomonas aeruginosa (MDRPA), the MIC of rifampicin is changed from 2 μg/mL to 0.125 μg/mL, the MIC of all-6-biguanide-beta-cyclodextrin is changed from more than 64mg/mL to 512 μg/mL, and the Fractional Inhibitory Concentration Index (FICI) obtained by the calculation of formulas 2 to 4 is less than 0.0705, exhibiting a synergistic effect.
As can be seen from FIG. 3 (right), when the total-6-Shan Guaji-beta-cyclodextrin and rifampicin act together on multi-drug resistant Pseudomonas aeruginosa (MDRPA), the MIC of rifampicin is changed from 2 mug/mL to 0.125 mug/mL, the MIC of the total-6-Shan Guaji-beta-cyclodextrin is changed from more than 64mg/mL to 1024 mug/mL, and the Fractional Inhibitory Concentration Index (FICI) obtained by the calculation of formulas 2 to 4 is less than 0.0785, which shows a synergistic effect.
As shown in fig. 3 (left): when the MIC of rifampicin is changed from 2 mug/mL to 1 mug/mL, the concentration of the required equal volume of the all-6-biguanide-beta-cyclodextrin is 64 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:64; when the MIC of rifampicin is changed from 2 mug/mL to 0.125 mug/mL, the concentration of the required equal volume of all-6-biguanide-beta-cyclodextrin is 512 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:4096;
as shown in fig. 3 (right): when the MIC of rifampicin is changed from 2 mug/mL to 0.125 mug/mL, the concentration of the required equal volume of all-6-Shan Guaji-beta-cyclodextrin is 1024 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:8196; when the MIC of rifampicin is changed from 2 mug/mL to 1 mug/mL, the concentration of the equal volume of all-6-Shan Guaji-beta-cyclodextrin is required to be 128 mug/mL, and the mass ratio of the antibiotic to the all-6-Shan Guaji-beta-cyclodextrin is 1:128.
FIG. 4 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin resistant multi-drug resistant Acinetobacter baumannii.
As can be seen from fig. 4 (left), when all-6-biguanide- β -cyclodextrin and rifampicin act together on multi-drug resistant acinetobacter baumannii (MDRAB), the MIC of rifampicin is changed from 4 μg/mL to 0.125 μg/mL, the MIC of all-6-biguanide- β -cyclodextrin is changed from more than 64mg/mL to 512 μg/mL, and the Fractional Inhibitory Concentration Index (FICI) obtained by the calculation of formulas 2 to 4 is less than 0.03925, exhibiting a synergistic effect.
As can be seen from FIG. 4 (right), the combination of all-6-Shan Guaji-beta-cyclodextrin and rifampicin acts on multi-drug resistant Acinetobacter baumannii (MDRAB), the MIC of rifampicin is changed from 1 μg/mL to 0.125 μg/mL, the MIC of all-6-Shan Guaji-beta-cyclodextrin is changed from more than 64mg/mL to 512 μg/mL, and the Fractional Inhibitory Concentration Index (FICI) obtained by calculation of formulas 2 to 4 is less than 0.133, exhibiting a synergistic effect.
As shown in fig. 4 (left): when the MIC of rifampicin is changed from 4 mug/mL to 2 mug/mL, the concentration of the required equal volume of all-6-biguanide-beta-cyclodextrin is 64 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:32; when the MIC of rifampicin is changed from 4 mug/mL to 0.125 mug/mL, the concentration of the required equal volume of all-6-holguanidyl-beta-cyclodextrin is 512 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanidino-beta-cyclodextrin is 1:4096;
as shown in fig. 4 (right): when the MIC of rifampicin is changed from 1 mug/mL to 0.5 mug/mL, the concentration of the required equal volume of all-6-Shan Guaji-beta-cyclodextrin is 64 mug/mL, and the mass ratio of the antibiotic to the all-6-Shan Guaji-beta-cyclodextrin is 1:128; when the MIC of rifampicin is changed from 1 mug/mL to 0.125 mug/mL, the concentration of the equal volume of all-6-Shan Guaji-beta-cyclodextrin is required to be 256 mug/mL, and the mass ratio of the antibiotic to the all-6-Shan Guaji-beta-cyclodextrin is 1:2048.
FIG. 5 is a graph showing the results of in vitro synergy experiments of whole guanidino substituted cyclodextrin and rifampicin against methicillin-resistant Staphylococcus aureus.
As can be seen from the left side of FIG. 5, when all-6-biguanide-beta-cyclodextrin and rifampicin act together on methicillin-resistant Staphylococcus aureus (MRSA), the MIC of rifampicin is changed from 4ng/mL to 0.25ng/mL, the MIC of all-6-biguanide-beta-cyclodextrin is changed from more than 64 μg/mL to 32 μg/mL, and a Fractional Inhibitory Concentration Index (FICI) of 0.5625 is obtained by calculation from formulas 2 to 4, exhibiting additive effects.
As can be seen from FIG. 5 (right), when all-6-Shan Guaji-beta-cyclodextrin and rifampicin act together on methicillin-resistant Staphylococcus aureus (MRSA), the MIC of rifampicin is changed from 4ng/mL to 1ng/mL, the MIC of all-6-Shan Guaji-beta-cyclodextrin is changed from more than 64 μg/mL to 32 μg/mL, and a Fractional Inhibitory Concentration Index (FICI) of less than 0.75 is calculated from formulas 2 to 4, exhibiting additive effects.
As shown in fig. 5 (left): when the MIC of rifampicin is changed from 4ng/mL to 2ng/mL, the concentration of the required equal volume of the all-6-biguanide-beta-cyclodextrin is 4 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:2000; when the MIC of rifampicin is changed from 4ng/mL to 0.25ng/mL, the concentration of the required equal volume of the all-6-biguanide-beta-cyclodextrin is 32 mug/mL, and the mass ratio of the antibiotic to the all-6-biguanide-beta-cyclodextrin is 1:128000
As shown in fig. 5 (right): when the MIC of rifampicin is changed from 4ng/mL to 2ng/mL, the concentration of the required equal volume of all-6-Shan Guaji-beta-cyclodextrin is 8 mug/mL, and the mass ratio of the antibiotic to the all-6-Shan Guaji-beta-cyclodextrin is 1:4000; when the MIC of rifampicin is changed from 4ng/mL to 1ng/mL, the concentration of the equal volume of all-6-Shan Guaji-beta-cyclodextrin is required to be 32 mug/mL, and the mass ratio of the antibiotic to the all-6-Shan Guaji-beta-cyclodextrin is 1:32000.
In conclusion, the full guanidine substituted cyclodextrin can restore the antibacterial sensitivity of rifampicin to multi-drug resistant pseudomonas aeruginosa, multi-drug resistant acinetobacter baumannii and methicillin-resistant staphylococcus aureus, can be used as an excellent antibiotic adjuvant to kill multi-drug resistant bacteria, and has higher application value.
EXAMPLE 4 application of hologuanidino-substituted Cyclodextrins in vivo synergistic experiments to killing of Lung infection in multidrug resistant bacteria
Female Balb/c mice (8 weeks old, 20 g) were tracheal injected with multidrug resistant Pseudomonas aeruginosa (MDRPA) (1×10) 8 CFU/mL) induced a mouse model of pneumonia. Infected mice were randomly divided into 3 groups and treated 2h after infection by tail vein injection administration, respectively. Three groups of mice were treated with PBS (100. Mu.L), rifampicin (10. Mu.g/kg), all-6-Shan Guaji-beta-cyclodextrin (10 mg/kg) +rifampicin (10. Mu.g/kg), respectively. Mice were euthanized 24h after infection. After collecting lung tissue homogenates from mice, the bacterial colony numbers were counted by plating on LB agar plates and direct dilution plating of alveolar lavage fluid (BALF).
FIG. 6 is a graph showing the results of in vivo synergy experiments of whole guanidyl substituted cyclodextrin and rifampicin resistant multidrug resistant Pseudomonas aeruginosa. Statistics of the number of bacteria in lung tissue and alveolar lavage fluid of mice after different treatments are counted, it can be seen from the graph that the number of MDRPA is found in the lung of mice treated with PBS or rifampicin, the number of bacteria in the lung of mice is significantly reduced after treatment with all-6-Shan Guaji-beta-cyclodextrin (10 mg/kg) +rifampicin (10 μg/kg), and the number of multidrug resistant pseudomonas aeruginosa (MDRPA) in the lung tissue is reduced by 100 times (left in FIG. 6) compared with the pure rifampicin treatment group or PBS treatment group by the combination of all-6-Shan Guaji-beta-cyclodextrin, so that the number of multidrug resistant pseudomonas aeruginosa (MDRPA) in the alveolar lavage fluid is reduced by 1000000 times (right in FIG. 6), and the bacteria are effectively cleared from the lung. The full-6-Shan Guaji-beta-cyclodextrin can still restore the use effect of the rifampicin in vivo as an antibiotic adjuvant, and overcomes the bacterial drug resistance.
EXAMPLE 5 application of full guanidino substituted cyclodextrins to Multi-drug resistant bacterial killing test
The Minimum Inhibitory Concentration (MIC) of whole guanidino-substituted cyclodextrin was tested against selected multi-drug resistant pseudomonas aeruginosa (MDRPA) using a mini broth dilution method.
Firstly, 70 mu L of Luria-Bertani (LB) culture solution is added into a 96-well plate, a prepared full-guanidino-substituted cyclodextrin solution with the concentration of 256mg/mL in advance is added into a first well, 70 mu L of mixed solution is added into a next well after uniform mixing, and half dilution is sequentially carried out except for a blank control group, so that the final concentration of materials in the well plate is 128 mg/mL and 64mg/mL respectively. Then adding an equal volume of diluted bacterial solution (2×10) of multidrug-resistant Pseudomonas aeruginosa (MDRPA) 5 CFU/mL), the final concentration of material in the well plate was 64, 32 up to 0.25mg/mL, respectively, and three replicates were set. Then cultured in a incubator at 37℃for 12 hours. The absorbance at 600nm is measured by using an enzyme-labeled instrument, and the calculation formula of the antibacterial efficiency (eta) is as follows: eta= [1- (A) 1 -A 2 )/(A 0 -A 3 )]Wherein A is 1 、A 2 、A 0 And A 3 The absorbance at 600nm was measured for each of LB medium containing the whole guanidyl-substituted cyclodextrin, LB medium containing the test strain, and LB medium alone. MIC quiltDefined as the lowest concentration of holohuanidino-substituted cyclodextrin where no significant bacterial growth was observed.
FIG. 7 is a graph showing the minimum inhibitory concentration of the whole guanidino-substituted cyclodextrin on multidrug resistant P.aeruginosa.
As can be seen from FIG. 7, when the concentration of the whole guanidyl substituted cyclodextrin is as high as 64mg/mL, the antibacterial rate of the whole-6-biguanidyl-beta-cyclodextrin and the whole-6-Shan Guaji-beta-cyclodextrin is about 50%, which shows that the whole guanidyl substituted cyclodextrin alone does not have the direct and efficient killing performance on drug-resistant bacteria, but can reduce the use amount of antibiotics and improve the sensitivity of the drug-resistant bacteria to the antibiotics when being combined with the antibiotics.
EXAMPLE 6 cytotoxicity test of full guanidino-substituted Cyclopaste
1. Mouse fibroblasts were cultured to log phase state.
2. And (3) paving: digesting cells from a culture flask by pancreatin, adding culture solution to blow off to obtain cell suspension, counting cells, taking a certain amount of cells to prepare 20 ten thousand/ml cell suspension, spreading 2 ten thousand cells per well on a 96-well plate, and culturing for 16 hours.
3. Adding materials. Preparing the whole guanidine group substituted cyclodextrin-DNA complex with different nitrogen-phosphorus ratios, and complexing the whole guanidine group substituted cyclodextrin with the DNA for half an hour.
4. The culture broth in the 96-well plate was removed and 100 μl of the formulated material was added to each well. The whole dead control group aspirates the culture broth and adds sterile water. Culturing for 4 hours.
5. The culture broth was then removed, gently rinsed twice with PBS, and the PBS was blotted clean with a 1mL and 100. Mu.L pipette, after which 100. Mu.L of MTT solution (5 mg MTT dissolved in 1mL PBS followed by 9mL serum-free culture broth) was added to each well.
6. Turning over after 4 hours. The toilet paper is paved on the table, the 96-well plate is gently and reversely buckled on the toilet paper, and the liquid in each hole is sucked out by the toilet paper. Then wash gently twice with PBS, remove clean PBS and add 100. Mu.L DMSO per well. Shake for 2 minutes using a shaker. The data is read.
FIG. 8 is a graph showing cytotoxicity of holguanidyl substituted cyclodextrins. As can be seen from fig. 8: the cell survival rate of the full-6-biguanide-beta-cyclodextrin and the full-6-guanidyl-beta-cyclodextrin is more than 80%, and the cell survival rate has no obvious toxicity, which indicates that the full-guanidyl substituted cyclodextrin has good biological safety.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. The application of the full-guanidino-substituted cyclodextrin in the aspect of being used as an antibiotic adjuvant is characterized in that the full-guanidino-substituted cyclodextrin is used as the antibiotic adjuvant, drug-resistant bacteria which have generated drug resistance to antibiotics are killed, the use amount of the antibiotics is reduced, the minimum inhibitory concentration of the antibiotics to the drug-resistant bacteria is reduced, the full-guanidino-substituted cyclodextrin is full-6-biguanide or full-6-Shan Guaji-substituted cyclodextrin, and the full-6-biguanide-beta-cyclodextrin has the general formula: (C) 7 H 13 N 5 O 4 ) n N=6, 7,8, the general formula of the all-6-Shan Guaji-beta-cyclodextrin is (C 7 H 13 N 3 O 4 ) n ，n＝6,7,8。
2. The use of a holguanidyl substituted ring paste according to claim 1 as an antibiotic adjuvant, wherein the mass ratio of antibiotic to holguanidyl substituted ring paste is 1:32 to 1:128000 when the holguanidyl substituted ring paste is used as an antibiotic adjuvant.
3. The use of a whole guanidino-substituted cyclodextrin as set forth in claim 1 as an antibiotic adjuvant, wherein the resistant bacteria are: drug-resistant pseudomonas aeruginosa, drug-resistant staphylococcus aureus, drug-resistant enterococcus faecium, drug-resistant klebsiella pneumoniae, drug-resistant acinetobacter baumannii, drug-resistant escherichia coli, drug-resistant brucella or drug-resistant tubercle bacillus.
4. The use of a whole guanidino-substituted cyclodextrin according to claim 1 as an antibiotic adjuvant, wherein the antibiotic is rifampin, tetracycline, streptomycin or doxycycline.
5. The use of a full guanidyl substituted ring paste according to any one of claims 1 to 4 as an antibiotic adjuvant, wherein the mass ratio of antibiotic to full guanidyl substituted ring paste used in combination is 1:64 to 1:8196 for multidrug resistant pseudomonas aeruginosa; the mass ratio of the antibiotic to the full guanidine-substituted ring paste used in a combined way is 1:32-1:4096 for the multi-drug resistant Acinetobacter baumannii, and the mass ratio of the antibiotic to the full guanidine-substituted ring paste used in a combined way is 1:2000-1:128000 for the methicillin-resistant staphylococcus aureus.
6. The use of a full guanidino-substituted ring paste according to claim 5 as an antibiotic adjuvant, wherein the mass ratio of antibiotic to full guanidino-substituted ring paste used in combination is 1:64-1:4096 and the mass ratio of antibiotic to Shan Guaji-substituted ring paste used in combination is 1:128-1:8196 for multidrug resistant pseudomonas aeruginosa; aiming at the multi-drug resistant Acinetobacter baumannii, the mass ratio of the antibiotic to the full guanidine-substituted ring paste is 1:32-1:4096, and the mass ratio of the antibiotic to the Shan Guaji-substituted ring paste is 1:128-1:2048; the mass ratio of the antibiotic to the full guanidine-substituted ring paste for the methicillin-resistant staphylococcus aureus is 1:2000-1:128000, and the mass ratio of the antibiotic to the full guanidine-substituted ring paste for the methicillin-resistant staphylococcus aureus is 1:4000-1:32000.
7. The use of a holguanidyl substituted ring paste according to claim 1 as an antibiotic adjuvant, wherein the holguanidyl substituted ring paste is prepared by the following steps:
1) Carrying out amination modification on cyclodextrin to obtain amino cyclodextrin;
2) Amino cyclodextrin reacts with dicyandiamide to obtain the full-6-biguanidino-beta-cyclodextrin or amino cyclodextrin reacts with 1H-pyrazole-1-formamidine hydrochloride to obtain the full-6-Shan Guaji-beta-cyclodextrin.
8. Use of a holguanidyl substituted ring paste according to claim 7 as an antibiotic adjuvant, characterized in that: step 1) substituting hydroxyl on the 6 th position of cyclodextrin with iodine, substituting iodine with azide, and reducing the azide to amino; the reaction conditions of step 1) are: the solvent of the reaction system is N, N-dimethylformamide, the reaction temperature is between room temperature and 70 ℃, and the reaction time is between 24 and 72 hours.
9. Use of a holguanidyl substituted ring paste according to claim 7 as an antibiotic adjuvant, characterized in that: the reaction conditions of the amino cyclodextrin and dicyandiamide in the step 2) are as follows: the mass ratio of the amino cyclodextrin to the dicyandiamide is 0.3-0.5, the reaction system is aqueous solution, the reaction temperature is 110-130 ℃, and the reaction time is 24-72 h.
10. Use of a full guanidino-substituted ring paste according to claim 7 as an antibiotic adjuvant, characterized in that the reaction conditions of the amino cyclodextrin of step 2) with 1H-pyrazole-1-carboxamidine hydrochloride are: the mass ratio of the amino cyclodextrin to the 1H-pyrazole-1-formamidine hydrochloride is 0.1-0.3, the solvent of the reaction system is N, N-dimethylformamide, the reaction temperature is 60-80 ℃, and the reaction time is 48-96H.
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