CN102626415A - Application of combination of tetracycline medicine and fluconazole in preparation of antifungal product, and product thereof - Google Patents
Application of combination of tetracycline medicine and fluconazole in preparation of antifungal product, and product thereof Download PDFInfo
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Abstract
The invention discloses an application of a combination of a tetracycline medicine and fluconazole (FLC) in the preparation of an antifungal product, and the product thereof, and concretely discloses a synergistic effect of three cell calcium regulators of a non-selective calcium channel retarding agent benidipine (BEN), a selective L-type calcium channel retarding agent nifedipine (NIF) and a calcium ion chelating agent ethylene diamine tetraacetic acid (EDTA) to the FLC and tetracycline antifungal medicines of doxycycline (DOX) and minocycline (MINO), and an application of the three cell calcium regulators in Candida albicans resisting, wherein a synergistic antifungal effect can be generated by the combined application of the FLC, the DOX and the MINO. Results confirm that the combined application of the FLC and the DOX/MINO can generate the synergistic antifungal effect, and has a substantial killing effect on drug-resistant Candida albicans; the BEN, the NIF and the EDTA can obviously enhance the combined antifungal effect of the FLC and the DOX/MINO to the Candida albicans, and can obviously reduce the lowest effective concentration during the combined application of the FLC, the MINO and the DOX; and the combined application of the three calcium regulators and the FLC can enhance the antifungal effect.
Description
Technical field
The present invention relates to medical technical field; Be specifically related to the application of tetracycline medication doxycycline and minocycline associating fluconazol in the preparation antifungal products, further relate to calcium channel blocker benidipine, nifedipine and calcium ion chelator EDTA associating fluorine chlorazol and fluconazol and the associating antifungic action of doxycycline/minocycline different sensitivity Candida albicans.The invention still further relates to the product that contains above-mentioned component or content.
Background technology
In recent years; Along with increasing of sickness rate such as malignant tumor, AIDS, autoimmune disease; Carrying out of technology such as intubation catheter and organ transplantation; Being widely used of broad ectrum antibiotic, 17-hydroxy-11-dehydrocorticosterone and immunosuppressant, the sickness rate of fungal infection sharply rise (Odds FC.Pathogenesis of Candida infections.J Am Acad Dermatol.1994,31:S2-S5).The particularly application of respirator and large number of biological material makes that (biofilm, BF) infection rate of relevant fungus constantly increases biomembrane.It is one of important mechanisms of clinical drug-resistant property that BF forms, and the clinical treatment of the relevant fungal infection of BF is difficulty very.Research shows; Behind AIDS patient's fungal infection in late period; Surpass 1/3rd drug resistances, to the azole drug resistant rate up to 65% (Saltanat H, Li H; Geng XH et al.Clinical distribution and drug resistance of candida infection.Journal of Xingjiang Medical University 2008,31 (8): 1058-1060.).New drug research and drug combination research all are to solve the drug-fast approach of fungus.And its early investment of drug combination is few, effect is remarkable, receives concern both domestic and external.Drug combination research comprises that two kinds of antifungal agent are united and non-antifungal agent and antifungal agent associating; Because the antifungal drug kind is limited; New antifungic action cost an arm and a leg and side effect obvious, make the Combined application research of non-antifungal drug and antifungal drug receive much concern.Fluconazol (FLC) is as safe, effective, inexpensive triazole antifungal agent thing, and clinical practice is very extensive, causes the fungus resistant rate constantly to rise.Therefore, become the focus of research with the associating antifungic action of FLC.This laboratory early-stage Study finds that minocycline (MINO) has collaborative antifungic action with FLC, and is particularly obvious to drug resistance Candida albicans effect.Find when studying the two synergistic mechanism that the change of the inside and outside calcium ion environment of cell can obviously influence collaborative antifungic action.Non-selective calcium ion channel blocker benidipine (BEN), selectivity L type calcium ion channel blocker nifedipine (NIF) and calcium ion chelator EDTA all produce stronger potentiation to its collaborative antifungic action.In addition; Further research shows; The doxycycline (DOX) that is all TCs share with FLC also has good collaborative antifungic action; And calcium regulator BEN, NIF and EDTA have potentiation equally to the collaborative antifungic action of DOX and FLC, and NIF, EDTA also have certain potentiation to FLC.
Before address, strengthening new drug development is the Critical policies of overriding resistance fungal infection.New drug development comprises the activity research of brand-new chemical compound, old medicine new role research.The discovery meeting of new mechanism, novel targets brings new hope to antifungal.Antifungal mechanism comprises at present: suppress the synthetic of fungal cell wall; The structure that suppresses the synthetic of fungal cell membrane main component ergosterol and directly act on ergosterol acts on fungal nucleic acid metabolism etc.Whether also exist other antifungal approach and mechanism to remain further to be studied.
Calcium ion is one of important courier's material of Candida albicans, participates in the information transmission and the regulation and control of a series of vital movements such as cell proliferation and differentiation, growth aging.Intracellular calcium overload is considered to the important pathology link of cell injury always.. discover; After the pathologic damage that the fungal cell causes drug effect; The homeostasis ability is main determining factor (Hameed S, Dhamgaye S, the Singh A that its drug resistance produces; Goswami SK, Prasad R (2011) Calcineurin Signaling and Membrane Lipid Homeostasis Regulates Iron Mediated MultiDrug Resistance Mechanisms in Candida albicans.PLoS ONE 6 (4): e18684.).Recent research report and our research find constantly that also it is relevant with antifungal that calcium is regulated path, are expected to become new antifungal mechanism.
Summary of the invention
The objective of the invention is for overcoming present fungal infection problem, the application in the preparation antifungal products of a kind of tetracycline medication doxycycline or minocycline Combined application fluconazol is provided; The present invention also provides calcium channel blocker benidipine, nifedipine and calcium ion chelator EDTA associating fluorine chlorazol and fluconazol and the application of doxycycline/minocycline in the preparation antifungal products.The invention still further relates to the product that contains above-mentioned component or content.
For realizing above-mentioned purpose, the present invention adopts following technical proposals:
The application in the preparation antifungal products of tetracycline medication doxycycline or minocycline Combined application fluconazol.
Said fungus is a Candida albicans.
The minimum inhibitory concentration of doxycycline and fluconazol is during Combined application: 16 μ g/ml and 4 μ g/ml; The minimum inhibitory concentration of minocycline and fluconazol is 16 μ g/ml and 2 μ g/ml.
The result shows, greater than 16 μ g/ml doxycycline and all effective greater than the fluconazol Combined application of 4 μ g/ml; Greater than 16 μ g/ml minocyclines and all effective greater than the fluconazol Combined application of 2 μ g/ml;
Add benidipine, nifedipine or metal ion chelation agent EDTA, plate streaking and liquid quantitative method all confirm to strengthen above-mentioned associating antifungic action.After adding benidipine, nifedipine or metal ion chelation agent EDTA, following concentration proportioning is effective:
Benidipine: doxycycline: fluconazol=8: 4: 2 (μ g/ml);
Benidipine: minocycline: fluconazol=8: 16: 1 (μ g/ml);
Nifedipine: doxycycline: fluconazol=8: 4: 1 (μ g/ml);
Nifedipine: minocycline: fluconazol=8: 8: 1 (μ g/ml);
EDTA: doxycycline: fluconazol=0.5mM: 4 μ g/ml: 1 μ g/ml;
EDTA: minocycline: fluconazol=0.5mM: 4 μ g/ml: 0.5 μ g/ml
When further the valid density of calcium regulator being estimated; Doxycycline is worked as in discovery and fluconazol concentration is 16 μ g/ml and 4 μ g/ml; When the minimum inhibitory concentration of minocycline and fluconazol is 16 μ g/ml and 2 μ g/ml; The valid density of the minimum calcium regulator that adds: benidipine is 4 μ g/ml, and nifedipine is 8 μ g/ml, and EDTA is 0.25mM.
The present invention also provides a kind of product that contains above-mentioned component or content.
The present invention adopts albicans cell research, utilizes plate streak and liquid quantitative method, estimates the associating antifungic action of different drug combination combinations respectively.Particular content is following:
The effect of A:FLC and DOX/MINO associating anti-candida albicans: MEC when adopting the liquid quantitative method to measure drug combination, and select the optimal drug combined concentration and estimate the effect of medication combined medication with the FICI method.
B: calcium regulator is united the influence of anti-candida albicans effect to FLC and DOX/MINO: experiment divides the blank group; FLC, DOX/MINO, the single group of using of BEN/NIF/EDTA; FLC and DOX/MIN or BEN/NIF/EDTA two medicine coupling groups; Four groups of FLC and DOX/MIN and BEN/NIF/EDTA three medicine coupling groups utilize plate streak observe to add behind the calcium regulator influence to the anti-white beads effect of each group.And utilize the liquid quantitative method to measure the associating MEC that calcium regulator share FLC and DOX/MINO.
C: tetracycline medication is clinical antibacterials commonly used, and is if can aspect antifungal, play a role, particularly to the treatment of antibacterial and fungus mixed infection, significant.This research shows that Tetracyclines associating FLC produces collaborative antifungic action, can enlarge its range of application.Calcium ion channel blocker is one type of depressor commonly used, can be divided into non-selective and selectivity calcium ion channel blocker two big classes.Enlarge the existing tangible feasibility of range of application and actual clinical meaning of calcium ion channel blocker class medicine.EDTA is the additive that can be used for food of the new authentication of FDA, and it can make its expanding application scope to the potentiation of antifungic action, has a good application prospect.
In a word, the present invention comprises the effect that FLC and DOX/MINO Combined application produce collaborative anti-candida albicans, and calcium regulator BEN, NIF and EDTA have potentiation to the associating antifungic action of FLC and FLC and tetracycline medication.Above-mentionedly be found to be the clinical drug combination that provides as treatment treatment of fungal infections scheme.In addition, the adding of calcium regulator can strengthen the antifungic action of medicine, and disclosing its synergism mechanism maybe be relevant with intracellular calcium, for the novel targets of studying the antifungal drug effect provides thinking.
The present invention compared with prior art has the following advantages and effect:
1. the present invention shows, when FLC and tetracycline medication DOX and MINO coupling, can produce collaborative antifungic action.To the drug-fast albicans strain of fluconazol (MIC＞256 μ g/ml); Drug combination can make its minimum inhibitory concentration obviously reduce; The FLC of the DOX of 16 μ g/ml and 4 μ g/ml share and the MINO of 16 μ g/ml and the FLC of 2 μ g/ml share the fungus that all can kill more than 80%; Concentration increases again, and effect is stronger.
2. the present invention shows, the non-selective calcium channel blocker benidipine of calcium regulator, selectivity L type calcium channel blocker nifedipine and calcium ion chelator EDTA all can strengthen the associating antifungic action that FLC and DOX/MINO share.When BEN concentration was 8 μ g/ml, NIF concentration was 8 μ g/ml, when EDTA concentration is 0.5mM above-mentioned drug combination combination was all had tangible associating antifungic action.Wherein, the adding of BEN, the concentration in the time of can making FLC and DOX coupling is reduced to 2 μ g/ml, 4 μ g/ml from 4 μ g/ml, 16 μ g/ml respectively; The adding of calcium ion chelator EDTA can make it reduce to 1 μ g/ml, 4 μ g/ml, and the adding of NIF can make it drop to 1 μ g/ml, 4 μ g/ml.In addition, BEN can make the concentration of FLC and MINO reduce to 1 μ g/ml, 8 μ g/ml from 2 μ g/ml, 16 μ g/ml; EDTA can make the MIC of drug combination reduce to 0.5 μ g/ml, 4 μ g/ml; NIF can make it drop to 1 μ g/ml, 8 μ g/ml.
3. the present invention shows, destroying the intracellular Ca2+ balance possibly be the mechanism of associating antifungic action, for the exploitation and the new usefulness of old medicine of new drug provides possible research direction.
4. tetracycline and calcium ion channel blocker extensive use clinically; It can enlarge its range of application to the potentiation of FLC antifungal, and reduces the MEC of FLC; Reduce the dosage of antifungal drug, thereby reduce the generation of the untoward reaction of medicine.
Description of drawings
Fig. 1: calcium regulator benidipine (BEN), nifedipine (NIF), calcium chelating agent EDTA is to the influence (48h growth result) of fluconazol (FLC) and doxycycline (DOX) anti-candida albicans (CA10, CA137, CA8, CA14) effect.The distribution of bacterial strain such as sketch map.
Fig. 2: calcium regulator benidipine (BEN), nifedipine (NIF), calcium chelating agent EDTA is to the influence (48h growth result) of fluconazol (FLC) and minocycline (MINO) anti-candida albicans (CA10, CA137, CA8, CA14) effect.The distribution of bacterial strain such as sketch map.
Fig. 3: rate of growth differential technique (Δ E method) is estimated non-selective calcium ion channel blocker benidipine (BEN) to the potentiation of fluconazol (FLC) with tetracycline medication (TC) drug combination.Vertical coordinate is represented the rate of growth difference that BEN share two medicines, and abscissa is represented different tetracycline medications and fluconazol associating.(FT: fluconazol and tetracycline medication share; FLC+MINO: fluconazol and minocycline share; FLC+DOX: fluconazol and doxycycline share).
Fig. 4: rate of growth differential technique (Δ E method) Evaluation and Selection property calcium ion channel blocker nifedipine (NIF) is to the potentiation to fluconazol (FLC) and tetracycline medication (TC) drug combination.Vertical coordinate is represented the rate of growth difference that NIF share two medicines, and abscissa is represented different tetracycline medications and fluconazol associating.(FT: fluconazol and tetracycline medication share; FLC+MINO: fluconazol and minocycline share; FLC+DOX: fluconazol and doxycycline share).
Fig. 5: rate of growth differential technique (Δ E method) is estimated calcium ion chelator EDTA to the potentiation to fluconazol (FLC) and tetracycline medication (TC) drug combination.Vertical coordinate is represented the rate of growth difference that EDTA share two medicines, and abscissa is represented different tetracycline medications and fluconazol associating.(FT: fluconazol and tetracycline medication share; FLC+MINO: fluconazol and minocycline share; FLC+DOX: fluconazol and doxycycline share).
Fig. 6: the Candida albicans inoculation precedence diagram of the different sensitivitys of four strains.
The specific embodiment
Through instantiation the present invention is further set forth below, should be noted that following explanation only is in order to explain the present invention, its content not to be limited.
Embodiment 1FLC and DOX/MINO associating antifungic action are measured
1. material
1.1 medicine and reagent
Fluconazol (fluconazole, FLC), medical sci-tech Development Co., Ltd is really created in Shandong;
Minocycline (minocycline, MINO), minocycline hydrochloride, Shandong huge rock letter bio tech ltd;
Doxycycline (Doxycycline, DOX), Shandong Province's drug identification institute;
Kerma (unit of kinetic energy) is praised the candidiasis chromogenic culture medium, the rich match in Zhengzhou biological engineering company limited;
The husky Borrow's agar of TTC-, sky, Hangzhou and microorganism reagent company limited;
Sodium hydroxide, state-run Shandong Dan County Organic Chemical Plant, lot number 940420;
Potassium dihydrogen phosphate, the new precious Fine Chemical Works in Shanghai, lot number 200602132.
Dimethyl sulfoxide (DMSO), Chinese Yao and Shun imports and exports company limited, lot number 031012;
RPMI 1640 raw material medicated powder, U.S. GIBCO company;
3-(N-morpholino) propane sulfonic acid (MOPS), Jinan friend Bioisystech Co., Ltd far away;
Menadione (Menadione), U.S. Sigma company;
XTT (the dimethoxy azoles is yellow), Nanjing optically-active Science and Technology Ltd.;
Lactated Ringer'S Solution (ringer's solution), Shandong Lukang Cisen Pharmaceutical Co., Ltd;
Acetone, Shanghai development chemical industry one factory, lot number 200209510;
The preparation of XTT-menadione solution: get XTT powder 0.0500g, be dissolved in the solution that the sterilized ringer's solution of 100ml is made into 0.5mg/ml, filter sterilization with 0.22 μ m filter membrane; Add the menadione acetone soln (get menadione 0.0860g and be dissolved in 5ml acetone) of the 10mmol/L of 10 μ l, making its final concentration is 1 μ mol/L, shakes up, and 2 ℃～8 ℃ keep in Dark Place.
Drug solution: fluconazol dissolves with sterile distilled water, is made into the storing solution of 2560 μ g/ml, filters packing; Fosfomycin is used sterilized water, and other drug is used dmso solution, is made into the storing solution of 6400 μ g/ml respectively, packing.All medicinal liquids are preserved in-20 ℃ of refrigerators, and are subsequent use.
PBS: measure 0.2mol/L potassium dihydrogen phosphate 50ml according to 2005 editions two ones of Chinese Pharmacopoeias, 0.2mol/L sodium hydroxide, 35ml are dissolved in the PBS buffer solution that promptly obtains pH 7.2 in the 200ml distilled water.121 ℃ of HTHP moist heat sterilization 20min cool off subsequent use.
RPMI 1640 liquid mediums: get RPMI 1640 and (contain L-glutaminate; Carbonated hydrogen sodium not) powder 2.08g; Add 10% glucose solution 40ml (containing sugared final concentration 2%) and MOPS powder 6.906g; Adding distil water transfers pH to be about 7.0 ± 0.1 at 22 ℃ of NaOH solution with 1mol/L to being about 200ml behind the mix homogeneously, faces with preceding usefulness 0.22 μ m composite fibre membrane filtration sterilization.
1.2 instrument
1.3 experimental strain
The Quality Control bacterial strain: Candida albicans ATCC10231, pharmacology teaching and research room of Shandong University is so kind as to give;
Experimental strain: provincial hospital, the isolating Candida albicans of Qianfo Mount hospital clinical;
Identification of strains: experiment was cultivated 48 hours down for 35 ℃ at the good candidiasis chromogenic culture medium of Kerma (unit of kinetic energy) with bacterial strain, and bacterium colony is green or emerald all bacterial strains are accredited as Candida albicans through Shandong Center for Disease Control & Prevention microbe research chamber with standard microorganism method again.
Bacterium liquid preparation: thaw under the Candida albicans room temperature that-20 ℃ are preserved down, be inoculated on the husky Borrow's agar culture medium of TTC-, cultivate 24～48h for 35 ℃, get well-developed single bacterium colony to inoculate once more, cultivate 24h, be in trophophase for 35 ℃ to guarantee bacterial strain.Choose some singlely than macrocolony, PBS is mixed with bacteria suspension, through the vortice vibration evenly the back with Chinese bacterial turbidity standard pipe than turbid, the adjustment sample cell is consistent with the standard pipe turbidity, the bacteria concentration of Candida albicans is about 4.5 * 10 at this moment
6CFU/ml, serial dilution promptly obtain work bacterium liquid, and carry out the concentration checking with count plate.
2. content and method
2.1 fluconazol and the effect of DOX/MINO associating anti-candida albicans are measured
Chessboard method according to CLSI M27-A2 scheme; Make it become 4 times of working concentrations with RPMI-1640 fluid medium dilute liquid medicine; Screening DOX and MINO and FLC concentration range of application; The final concentration that is DOX and MINO is respectively 32～0.5 μ g/ml and 64～1 μ g/ml, and FLC is 512～0.5 μ g/ml.Draw azole medicinal liquid 50 μ l by concentration order from high to low; Add the 12nd～2 dull and stereotyped row of 96 holes respectively; Draw medicinal liquid 50 μ l such as minocycline by concentration order from high to low; It is capable to add the dull and stereotyped the A～G in 96 holes respectively, and each hole adds 100 μ l bacterium liquid more respectively except that A12, and supply with the RPMI-1640 culture fluid in the hole of all the other less than 200 μ l.Wherein H1 is a growth control, only contains not drug of bacterium liquid, and A12 is a blank, only contains medicinal liquid and does not contain bacterium liquid.With 96 hole flat boards put in 35 ℃ of constant incubators cultivate 48h after, with measure and write down the result after the XTT load with ELIASA.All test triplicate.
2.2 evaluation methodology and result judge
The theoretical basic thought of Loewe additivity (LA) thinks that medicine can not interact with itself, so the concentration (equivalent site) that medicine list usefulness or coupling produce identical drug effect is compared.Its analytical method mark Mlc index method (fractional inhibitory concentration index, FICI), explain as follows:
∑FIC＝FIC
A+FIC
B＝C
A/MIC
A+C
B/MIC
B
MIC
AAnd MIC
BBe respectively medicine A and the B minimal inhibitory concentration of single time spent, C
AWith C
BSeparately concentration when reaching identical drug effect when being two medicine couplings.FICI＞4 are antagonism, and FICI is addition or irrelevant effect between 0.5 and 4, and FICI≤0.5 is defined as synergism.
3. result
3.1 the associating antifungic action result of fluconazol and antibacterials
3.1.1 minimum effective bacteria concentration of fluconazol and antibacterials associating
The computational methods of conk percent are in each hole:
Conk percent=(each hole OD value-blank hole OD value)/growth control hole OD value
Calculate each hole conk percent in the flat board by above-mentioned formula, getting the minimum coupling drug level that can suppress conk 80% is the interpretation terminal point, if the conk rate does not equal 20% just, getting immediate with it medication combined hole is the interpretation terminal point.
The associating antifungic action of FLC and DOX/MINO, not obvious concerning sensitive strain, be mostly irrelevant, and, then present strong synergism persister.Wherein the growth percent experimental result in the persister CA10 flat board is as shown in table 1.
Table .1. representes the drug combination anti-candida albicans CA of FLC and DOX/MINO with chessboard
10Growth percentage rate (the medicine the best use of combination that converts with the FICI method marks with Lycoperdon polymorphum Vitt).
4. conclusion:
Less and the present situation of drug resistance phenomenon occurs in the face of antifungal drug in the clinical practice; Developing novel antifungal drug is one of approach that addresses this problem, and to increase fungus also be a kind ofly to select preferably to the sensitivity of medicine to the mode through drug combination simultaneously.Existing bibliographical information, though TCs has the effect of anti-candida albicans, minocycline, doxycycline antifungal activity very a little less than.But the DOX and the MINO of this experiment proof low concentration can strengthen the effect of fluconazol to the Candida albicans persister; Played mutual synergic effect; And can eliminate " hangover " phenomenon (at the drug level that surpasses the MIC value; Candida albicans still has and continues a spot of growth), but its mechanism of action remains further to be studied.
Embodiment 2 calcium regulators are to the influence of FLC and DOX/MINO antifungic action
1. material
1.1. medicine and reagent
Benidipine (KW-3049, BEN), China pharmaceutcal corporation, Ltd;
EDTA, Chinese Shanghai reagent one factory;
Nifedipine (nifedipine, NIF), Shandong Province's medicine is identified institute;
Doxycycline (Doxycycline, DOX), Chinese medicine is identified institute;
Mother liquid medicine: following all medicinal liquids are preserved subsequent use in-20 ℃ of refrigerators.
Benidipine is made into the storing solution of 2560 μ g/ml with DMSO
Minocycline is made into the storing solution of 6400 μ g/ml with DMSO
Fluconazol is made into the storing solution of 2560 μ g/ml with sterile distilled water
Doxycycline is made into the storing solution of 6400 μ g/ml with sterile distilled water
Nifedipine is made into the storing solution of 6400 μ g/ml with sterile distilled water
EDTA is made into the storing solution of 0.5M with sterile distilled water
Yeast extract-peptone-glucose (YPD) agar culture medium: glucose 20g, peptone 20g, yeast extract 10g, agar powder 20g uses the 1000ml dissolved in distilled water, stirs back 121 ℃ of sterilization 20min, cools off back 4 ℃ of refrigerators and preserves subsequent use.
PBS: measure 0.2mol/L potassium dihydrogen phosphate 50ml according to 2005 editions two ones of Chinese Pharmacopoeias, 0.2mol/L sodium hydroxide, 35ml are dissolved in the PBS buffer solution that promptly obtains pH 7.2 in the 200ml distilled water.121 ℃ of HTHP moist heat sterilization 20min cool off subsequent use.
1.2. instrument
1.3. experimental strain
Through Candida albicans persister CA10, the CA137 that identifies; Sensitive strain CA14, CA8.
2. method
2.1. the preparation of bacterium liquid
The different Candida albicans CA8 of well-grown four strain varying sensitivities after picking goes down to posterity respectively; CA10, CA14, the single bacterium colony of CA137; Be mixed with bacteria suspension with 0.9% normal saline; Behind vortice vibration mixing with Chinese bacterial turbidity standard pipe than turbid, when adjustment sample cell and standard pipe turbidity basically identical, the bacteria concentration of Candida albicans is about 4.5 * 10
6CFU/ml.The bacteria suspension of each Candida albicans strain of this concentration is diluted 10 times with sterile saline, become about 4.5 * 10
5The work bacterium liquid of CFU/ml concentration,
2.2. the preparation that pastille is dull and stereotyped
The YPD Agr culture medium is taken out in 4 ℃ of refrigerators, and slow heating and melting is measured about 16ml with graduated cylinder after the cold slightly and is poured in the glass culture dish in microwave oven.Each mother liquid medicine is diluted with sterile purified water; Draw by a certain percentage, the final concentration that makes interior medicine of 16ml culture medium and/or calcium regulate reagent can reach minocycline 8 μ g/ml, the about 2 μ g/ml of fluconazol; DOX 8 μ g/ml; The about 8 μ g/ml of benidipine, the about 0.5Mm of EDTA, the about 8 μ g/ml of nifedipine.The medicine of above-mentioned sucking-off or reagent solution are joined temperature reduce in the plate that contains liquid YPD Agr culture medium about 35～40 ℃, form the pastille system, jiggle mixing, level leaves standstill, after solidifying pastille is dull and stereotyped.
2.3. plate streaking and cultivation
The cotton swab of picking head size thickness homogeneous is reduced to room temperature and is taken out behind 160 ℃ of dry heat sterilization 2h meticulously.During use, its head is soaked about 4min in the work bacterium liquid of 3ml, make the cotton swab complete wetting.Before the line bacterium liquid is shaken with the cotton swab spiral, the fungal cell is adhered on cotton swab as far as possible uniformly, thereby dip in the bacterium liquid of respectively working that takes out the concentration basically identical.With the cotton swab evenly streak inoculation in the dull and stereotyped specific region of each pastille of carrying disease germs, the Candida albicans inoculation order of the different sensitivitys of four strains is as shown in Figure 6.At last with flat board in 35 ℃ of constant temperature culture, observe the strain growth situation behind the 48h.
3. result
3.1 calcium regulator is to the influence of FLC and DOX antifungic action
As shown in fig. 1; FLC is single with the quantity that can reduce conk; But DOX (16 μ g/ml) and calcium channel blocker BEN (8 μ g/ml), NIF (8 μ g/ml), singly using of EDTA (0.5mM) do not have macroscopic growth or inhibitory action to conk.As the DOX of 16 μ g/ml, NIF (8 μ g/ml), EDTA (0.5mM), all to the oidiomycetic growth inhibited effect of white, and effect obviously is better than, and FLC is single to be used during BEN (8 μ g/ml) and 2 μ g/mlFLC associating.Yet, when calcium regulator and DOX and three kinds of calcium regulators share, fungus is not had the obvious suppression effect.When calcium regulator and FLC-DOX share, all making of BEN, NIF and EDTA almost do not have macroscopic strain growth on the flat board, and the effect that suppresses conk is stronger, united with any two medicines and compared the effect that suppresses conk and significantly strengthen.
3.2 calcium regulator is to the influence of FLC and MINO antifungic action
Calcium regulator is similar with the result of FLC-DOX to the oidiomycetic effect of white, promptly uses calcium regulator and MINO to almost not influence of fungi growth separately, MINO single with and and calcium regulator share and all do not have tangible anti-candida albicans effect; When list was used FLC, the growth of visible Candida albicans received slight inhibition, with BEN, NIF, antifungic action strengthened to some extent when EDTA share.The adding of MINO has strengthened FLC to the oidiomycetic inhibitory action of white; Calcium regulator is as shown in Figure 2 to MINO and the symphyogenetic influence of azole drug: all making of BEN, NIF and EDTA almost do not have macroscopic strain growth on the flat board, unites the effect of comparing the inhibition conk with any two medicines and significantly strengthens.
4. discuss and conclusion
It is more obvious when acting on fluconazol concentration＞4 μ g/ml that TCs minocycline and doxycycline and fluconazol Combined application dialogue be oidiomycetic; To sensitive organism (CA8, effect CA14) is better, almost has only fastbacteria (CA10 on the flat board; CA137) growth (result does not show); But our research purpose is that various calcium are regulated the effect of factor to drug combination, therefore, draws when concentration is 2 μ g/ml through repeatedly testing the condition of groping; Sensitive organism and fastbacteria all have growth, are beneficial to each effect of factors of research.The adding of calcium regulator can increase the antifungic action effect of drug combination greatly, but concrete concentration and the effect under liquid environment are not bright, needs further to confirm its effect, and it is strong and weak to estimate potentiation, analyzes its possible mechanism.
Embodiment 3 calcium regulators are to the quantitative assay of drug combination potentiation
1. material
1.1 medicine and reagent
Benidipine (KW-3049, BEN), China pharmaceutcal corporation, Ltd;
EDTA, Chinese Shanghai reagent one factory;
Nifedipine (nifedipine, NIF), the Central China University of Science and Technology is so kind as to give the pharmacological evaluation chamber;
Doxycycline (Doxycycline, DOX), Chinese medicine is identified institute;
Mother liquid medicine: following all medicinal liquids are preserved subsequent use in-20 ℃ of refrigerators.
Benidipine is made into the storing solution of 2560 μ g/ml with DMSO
Minocycline is made into the storing solution of 6400 μ g/ml with DMSO
Fluconazol is made into the storing solution of 2560 μ g/ml with sterile distilled water
Doxycycline is made into the storing solution of 6400 μ g/ml with sterile distilled water
Nifedipine is made into the storing solution of 6400 μ g/ml with sterile distilled water
EDTA is made into the storing solution of 0.5M with sterile distilled water
Kerma (unit of kinetic energy) is praised the candidiasis chromogenic culture medium, the rich match in Zhengzhou biological engineering company limited;
The husky Borrow's agar of TTC-, sky, Hangzhou and microorganism reagent company limited;
Sodium hydroxide, state-run Shandong Dan County Organic Chemical Plant, lot number 940420;
Potassium dihydrogen phosphate, the new precious Fine Chemical Works in Shanghai, lot number 200602132.
Dimethyl sulfoxide (DMSO), Chinese Yao and Shun imports and exports company limited, lot number 031012;
RPMI 1640 raw material medicated powder, U.S. GIBCO company;
3-(N-morpholino) propane sulfonic acid (MOPS), Jinan friend Bioisystech Co., Ltd far away;
Menadione (Menadione), U.S. Sigma company;
XTT (the dimethoxy azoles is yellow), Nanjing optically-active Science and Technology Ltd.;
Lactated Ringer'S Solution (ringer's solution), Shandong Lukang Cisen Pharmaceutical Co., Ltd;
Acetone, Shanghai development chemical industry one factory, lot number 200209510;
The preparation of XTT-menadione solution: get XTT powder 0.0500g, be dissolved in the solution that the sterilized ringer's solution of 100ml is made into 0.5mg/ml, filter sterilization with 0.22 μ m filter membrane; Add the menadione acetone soln (get menadione 0.0860g and be dissolved in 5ml acetone) of the 10mmol/L of 10 μ l, making its final concentration is 1 μ mol/L, shakes up, and 2 ℃～8 ℃ keep in Dark Place.
PBS: measure 0.2mol/L potassium dihydrogen phosphate 50ml according to 2005 editions two ones of Chinese Pharmacopoeias, 0.2mol/L sodium hydroxide, 35ml are dissolved in the PBS buffer solution that promptly obtains pH 7.2 in the 200ml distilled water.121 ℃ of HTHP moist heat sterilization 20min cool off subsequent use.
RPMI 1640 liquid mediums: get RPMI 1640 and (contain L-glutaminate; Carbonated hydrogen sodium not) powder 2.08g; Add 10% glucose solution 40ml (containing sugared final concentration 2%) and MOPS powder 6.906g; Adding distil water transfers pH to be about 7.0 ± 0.1 at 22 ℃ of NaOH solution with 1mol/L to being about 200ml behind the mix homogeneously, faces with preceding usefulness 0.22 μ m composite fibre membrane filtration sterilization.
1.2 instrument
1.3 experimental strain
The Quality Control bacterial strain: Candida albicans ATCC10231, pharmacology teaching and research room of Shandong University is so kind as to give;
Experimental strain: provincial hospital, the isolating Candida albicans of Qianfo Mount hospital clinical;
Identification of strains: experiment was cultivated 48 hours down for 35 ℃ at the good candidiasis chromogenic culture medium of Kerma (unit of kinetic energy) with bacterial strain, and bacterium colony is green or emerald all bacterial strains are accredited as Candida albicans through Shandong Center for Disease Control & Prevention microbe research chamber with standard microorganism method again.
Bacterium liquid preparation: thaw under the Candida albicans room temperature that-20 ℃ are preserved down, be inoculated on the husky Borrow's agar culture medium of TTC-, cultivate 24～48h for 35 ℃, get well-developed single bacterium colony to inoculate once more, cultivate 24h, be in trophophase for 35 ℃ to guarantee bacterial strain.Choose some singlely than macrocolony, PBS is mixed with bacteria suspension, through the vortice vibration evenly the back with Chinese bacterial turbidity standard pipe than turbid, the adjustment sample cell is consistent with the standard pipe turbidity, the bacteria concentration of Candida albicans is about 4.5 * 10 at this moment
6CFU/ml, serial dilution promptly obtain work bacterium liquid, and carry out the concentration checking with count plate.
2. experimental technique
2.1BEN mensuration to associating antifungic action MEC
Select variable concentrations BEN (4 μ g/ml, 8 μ g/ml, 16 μ g/ml), EDTA (0.25mM, 0.5Mm, 1mM) and NIF (4 μ g/ml, 8 μ g/ml, 16 μ g/ml).
Chessboard method according to CLSI M27-A2 scheme; Make it become 4 times of working concentrations with RPMI-1640 fluid medium dilute liquid medicine; Select associating antifungic action the strongest antibacterials and fluconazol concentration ranges of application such as screening minocycline for use; The final concentration that is MINO and DOX is respectively 64～1 μ g/ml and 32～0.5 μ g/ml, and FLC is 512～0.5 μ g/ml.Draw azole medicinal liquid 50 μ l by concentration order from high to low; Add the 12nd～2 dull and stereotyped row of 96 holes respectively; Draw medicinal liquid 50 μ l such as minocycline by concentration order from high to low; It is capable to add the dull and stereotyped the A～G in 96 holes respectively, and each hole adds the NIF solution of 50 μ l bacterium liquid and 50 μ l BEN solution or 50 μ l or the EDTA solution of 50 μ l more respectively except that A12.Supply with the RPMI-1640 culture fluid in the hole of all the other less than 200 μ l.Wherein H1 is a growth control, only contains not drug of bacterium liquid, and A12 is a blank, only contains medicinal liquid and does not contain bacterium liquid.With 96 hole flat boards put in 35 ℃ of constant incubators cultivate 48h after, with measure and write down the result after the XTT load with ELIASA.All test triplicate.
2.2 the evaluation methodology of potentiation and result judge
This experiment is chosen the minimum concentration of effectively share that FLC and tetracycline medication share as experimental subject; Estimate interaction in vitro result and the intensity of calcium regulator with XTT method observational method to medication combined application; Confirm the MIC value of calcium regulator effect, and the FICI value that the calculating calcium regulator adds back FLC and tetracycline medication is to confirm whether calcium regulator has the MEC that can reduce FLC and DOX and MINO drug combination.And judge that through BI Theoretical Calculation Δ E calcium regulator acts on the influence of pharmaceutically-active critical point.
The BI theory is described with following formula: when Ei=EA * EB, Ei are medicine A and B coupling under no interactional hypothesis theoretic conk percent, EA and EB are respectively two kinds of medicines actual conk percent when using separately.Drug interaction is estimated with Δ E value, i.e. percentile theoretical value of conk and test value poor under each concentration of medicine.Because the associating drug sensitive experiment is to use the Microdilution flat board to cooperatively interact with two-fold dilution's amount of each medicine, therefore group is medication combined can obtain a Δ E value to each.When the meansigma methods of Δ E for just, and its 95% confidence interval do not comprise at 0 o'clock, thinks that two medicines are synergism; When mean difference for negative and its 95% confidence interval do not comprise 0, be antagonism.
3. result
3.1FICI method is estimated the potentiation of calcium regulator
For three repeated experiments, in order to explain the result of gained data, Odds suggestion FICI≤0.5 is defined as synergism, and FICI＞4 are antagonism, and 4＜FICI≤0.5 is irrelevant effect.It is visible to measure the result by table 2, when DOX/MINO and fluconazol Combined application, to the action effect of sensitive organism and fastbacteria equal＜0.5, present synergism; The adding of calcium regulator has increased the synergism of drug combination in various degree, and the FICI value all reduces.
Table 2 comments calcium regulator that the drug combination antifungic action influenced Table 1:The results of drug effect are evaluated by FICI and Δ E method with the FICI method
3.2 rate of growth differential technique (Δ E method) is estimated the potentiation of variable concentrations calcium regulator to drug combination
This experimental section is chosen the best use of point of non-drug combination, observes the influence of variable concentrations calcium regulator to the anti-mycotic efficiency of this drug regimen.For three repeated experiments, when the average of Δ E for just, and its 95% confidence interval do not comprise at 0 o'clock, drug effect is a synergism; When the average of Δ E for negative, and its 95% confidence interval do not comprise at 0 o'clock, drug effect is the remarkable antagonism of statistics; Other is the irrelevant effect of Bliss.Choosing minimum drug effect concentration point and two drug effect combinations of upstream and downstream thereof of the combination of experiment two Chinese medicines, is the X axle with Δ E value, observes the potentiation of calcium regulator to medicine.
3.2.1BEN influence to the drug combination antifungic action
As shown in Figure 3, when adding BEN concentration less than 4 μ g/ml, BEN is also not obvious to the potentiation of the collaborative antifungic action of drug combination; When BEN concentration＞4 μ g/ml, along with the potentiation of the increase medicine of BEN concentration strengthens, when BEN concentration was 16 μ g/ml, this medication combination can be killed all funguses basically.
3.2.2NIF influence to the drug combination antifungic action
As shown in Figure 4, when adding NIF concentration less than 4 μ g/ml, NIF is also not obvious to the potentiation of the collaborative antifungic action of drug combination; When BEN concentration＞4 μ g/ml, along with the potentiation of the increase medicine of BEN concentration strengthens, when BEN concentration was 16 μ g/ml, this medication combination can be killed all funguses basically.
3.2.3EDTA influence to the drug combination antifungic action
As shown in Figure 5, when adding EDTA concentration less than 0.25mM, EDTA is also not obvious to the potentiation of the collaborative antifungic action of drug combination; When EDTA concentration is 0.25mM, can increase the antifungic action of medicine greatly, its bacteriostasis rate is obviously increased, Δ E value raises obviously; When EDTA concentration＞0.5mM, the adding of EDTA can make drug combination when this concentration combination, kill all funguses.
4. discuss
Confirm all that with FLCI method and Δ E method the calcium regulator distich share medical instrument potentiation is arranged, the adding of calcium regulator can make the activity of drug combination reduce.Constantly rise in present fungus resistant rate, medicine upgrades under the severe slowly situation, and drug combination is control infection effectively.Calcium ion channel blocker is as clinical depressor commonly used; It strengthens the discovery of FLC and DOX or MINO associating antifungic action; Can solution be provided for clinical fungus drug resistance problem; Enlarge the range of application of calcium ion channel blocker, and new research direction is provided for the research and development of antifungal drug.
Claims (7)
1. tetracycline medication doxycycline or the minocycline Combined application fluconazol application in the preparation antifungal products.
2. application as claimed in claim 1 is characterized in that, said fungus is a Candida albicans.
3. application as claimed in claim 1 is characterized in that, the minimum inhibitory concentration of doxycycline and fluconazol is during Combined application: 16 μ g/ml and 4 μ g/ml; The minimum inhibitory concentration of minocycline and fluconazol is 16 μ g/ml and 2 μ g/ml.
4. application as claimed in claim 1 is characterized in that, also adds benidipine, nifedipine or metal ion chelation agent EDTA.
5. application as claimed in claim 4 is characterized in that, after adding benidipine, nifedipine or metal ion chelation agent EDTA, its concentration proportioning is:
Benidipine: doxycycline: fluconazol=8:4:2 (μ g/ml);
Benidipine: minocycline: fluconazol=8:16:1 (μ g/ml);
Nifedipine: doxycycline: fluconazol=8:4:1 (μ g/ml);
Nifedipine: minocycline: fluconazol=8:8:1 (μ g/ml);
EDTA: doxycycline: fluconazol=0.5mM:4 μ g/ml:1 μ g/ml;
EDTA: minocycline: fluconazol=0.5mM:4 μ g/ml:0.5 μ g/ml.
6. application as claimed in claim 4; It is characterized in that; When doxycycline and fluconazol concentration are the minimum inhibitory concentration of 16 μ g/ml and 4 μ g/ml or minocycline and fluconazol when being 16 μ g/ml and 2 μ g/ml, the valid density of the minimum calcium regulator of adding: benidipine is that 4 μ g/ml, nifedipine are that 8 μ g/ml or EDTA are 0.25mM.
7. an antifungal products is characterized by and contains each said component or content among the claim 1-6.
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| CN105497026A (en) * | 2015-12-14 | 2016-04-20 | 山东省千佛山医院 | Application of composition of benzenebutanoic acid and triazole antifungal drug and antifungal product |
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