CN105663126A

CN105663126A - Antifungal product combining ambroxol hydrochloride with fluconazole and application thereof

Info

Publication number: CN105663126A
Application number: CN201610186067.8A
Authority: CN
Inventors: 孙淑娟; 李秀云; 张才擎; 韩毅; 崔学艳
Original assignee: Shandong Qianfoshan Hospital
Current assignee: Shandong Qianfoshan Hospital
Priority date: 2016-03-29
Filing date: 2016-03-29
Publication date: 2016-06-15
Anticipated expiration: 2036-03-29
Also published as: CN105663126B

Abstract

The invention discloses an antifungal product combining ambroxol hydrochloride with fluconazole and application thereof and belongs to the technical field of medicine. Static and dynamic combined antifungal effect, on medicine-resistant Candida albicans, of combined use of fluconazole and ambroxol hydrochloride is studied through various methods. An effective concentration proportion of fluconazole to ambroxol hydrochloride when ambroxol hydrochloride and fluconazole are combined for application is 1:64 (ug/mL), and lowest antibacterial concentrations are 128 ug/mL and 2 ug/mL respectively. When ambroxol hydrochloride is combined with fluconazole, antibacterial activity, to medicine-resistant Candida albicans, of fluconazole can be improved, synergistic antifungal effect can be generated, medicine resistance, to fluconazole, of medicine-resistant Candida albicans can be reversed, and a studying direction is provided for development of new drugs and new application of old drugs.

Description

The antifungal products of ambroxol hydrochloride associating fluconazol and application thereof

Technical field

The present invention relates to pharmaceutical technology field, be specifically related to antifungal products and the application thereof of ambroxol hydrochloride associating fluconazol.

Background technology

In recent years, along with increasing and the extensive use of extensive pedigree antibiotic of acquired immune deficiency syndrome (AIDS) (AIDS) patient, carrying out of the medical skill such as organ transplantation technique and microcatheter technology, sickness rate and the case fatality rate of invasive infections with fungi rise year by year, particularly aspergillus and candidal infection, the survival rate of patient is respectively less than 30% and 70%. Although clinical threat is not had the threat that aspergillin infection causes big by monilial infection, but owing to this kind of fungus easily occurs that drug efflux strengthens and gene Target alterations (such as ERG11) etc., making the candidiasis to triazole antifungal agent thing is sensitive drug resistance constantly occur, this brings great challenge to clinical anti-fungal infection. Data show, after AIDS patient later infections fungus, more than 1/3rd drug resistances, to azole drug resistant rate especially up to 65%. Wherein candidiasis especially Candida albicans (Candidaalbicans, CA), is the common separation bacterium of respiratory system, bloodstream infection, digestive system, fungal Infections of Urinary System. The data controlling tissue according to nosocomial infection of the U.S. shows, CA is the 4th and causes the pathogenic microorganism of bloodstream infection in institute, is the pathogenic bacterium that mortality rate is the highest, and mortality rate may be up to 40%. The high mortality of CA bloodstream infection is also relevant with the continuous appearance of its drug resistance phenomenon. New drug research and drug combination research are all the approach solving antifungal agent resistance. Owing to drug combination early investment is few, effect notable, it is subject to the extensive concern of domestic and international researcher. Drug combination research includes two kinds of antifungal agent associatings and non-antifungal agent is combined with antifungal agent, owing to current useful clinically antifungal drug kind is limited, and mostly expensive, toxic and side effects is obvious so that the use in conjunction research of non-antifungal drug and antifungal drug receives much concern. Azole drug in recent years, especially fluconazol, although extensive use achieve good clinical effectiveness, but the appearance of thing followed persister makes clinical treatment become thorny, and this makes to overcome Candida albicans that the resistance problems of fluconazol is become study hotspot.

Ambroxol hydrochloride (Ambroxolhydrochloride, ABH) is a kind of safely, effectively medicine treating respiratory system disease, is widely used in the reducing phlegm of common respiratory tract disease, expectoration. clinical application report is had to find in recent years, ambroxol hydrochloride can play collaborative antibacterial actions with antibiotic, the treatment time of respiratory system infection can be shortened, reduce complication rate and case fatality rate, it is suitable for the age broad, can be widely used for various acute and chronic pulmonary disease, respiratory distress syndrome, there is reduction sputum viscosity, improve the effect of patient's expectoration function and pulmonary function, determined curative effect, patient generally can tolerate very well. at present, ABH has the chance (Sun Hongshuan with antifungal drug use in conjunction clinically, narrow eyes into a slit spring city, Ma Hongfang, Chen Hejun, plant Pharmaceutical Care [J] .2015 of valuable .1 example Aged Patients With Lung bacteria mixed fungi infected patient, 12 (3): 182-185.), additionally, finding in clinical treatment that ambroxol injection and antibacterials are share can make antibacterials raise at the distributed density of lung tissue, strengthen the effect (Wang Yongli of antibacterials, Cao Yongning. ambroxol injection treatment 56 cases of mycoplasmal pneumonia in children [J]. China's Pharmaceutical, 2014, 23 (19): 97-98.). for the Effect study of the coupling anti-candida albicans of ABH and FLC, there is no research report at present, there is wide Research Prospects.

Summary of the invention

The invention aims to overcome current antifungal agent resistance problem, it is provided that the antifungal products of a kind of ambroxol hydrochloride associating fluconazol and application thereof.

To achieve these goals, the present invention adopts the following technical scheme that

The associating fluconazol application in preparing antifungal products of a kind of ambroxol hydrochloride.

Preferred: described product is medicine.

Preferred: described fungus is Candida albicans.

Preferred: valid density proportioning when described ambroxol hydrochloride and fluconazole application: fluconazol: ambroxol hydrochloride=1:64.

During described application, the minimum inhibitory concentration of ambroxol hydrochloride is: 128 μ g/mL.

During described application, the minimum inhibitory concentration of fluconazol is: 2 μ g/mL.

A kind of antifungal products, with ambroxol hydrochloride and fluconazol for main active.

Preferred: the valid density proportioning of described ambroxol hydrochloride and fluconazol is fluconazol: ambroxol hydrochloride=1:64.

The least concentration of described ambroxol hydrochloride is: 128 μ g/mL.

The least concentration of described fluconazol is: 2 μ g/mL are it is shown that the ambroxol hydrochloride more than 128 μ g/mL has collaborative overriding resistance Candida albicans effect with the fluconazole application more than 2 μ g/mL.

A kind of antifungal preparation, is made up of ambroxol hydrochloride and fluconazol and pharmaceutically acceptable adjuvant. In preparation provided by the invention, pharmaceutically acceptable adjuvant is the customary adjuvant in pharmaceutical preparation, thinks that feasible customary adjuvant is all within protection scope of the present invention for those skilled in the art, and the present invention does not limit at this.

It is a discovery of the invention that ABH can play associating antifungic action with antifungal drug in vitro, it can be seen that, both collaborative antifungic actions are not limited only to ABH makes antibacterials raise this mechanism of action at the distributed density of lung tissue.

The present invention adopts drug resistance albicans cell to study, and utilizes chessboard broth microdilution antifungal susceptibility test, evaluates the associating antifungic action of different drug combination combination respectively. Particular content is as follows:

A:ABH and FLC combines overriding resistance CA effect: adopt chessboard broth microdilution antifungal susceptibility test to measure minimal effective concentration during drug combination, selects optimal drug combined concentration with FICI method and evaluates the effect of combination therapies.Additionally, be the drug combination degree to drug resistance CA effect that more intuitively shows, associating drug sensitive detection data grown percentage difference value model (Δ E) and has carried out further analysis.

B:ABH in Clinical practice consumption greatly, safely, effectively, if can play a role in antifungal, particularly to drug resistance fungal infect treatment, significant. The study show that, ABH use in conjunction FLC can produce collaborative overriding resistance CA effect, can expand its range of application, have a good application prospect.

In a word, the present invention comprises the effect of the collaborative overriding resistance CA of ABH and FLC use in conjunction generation. Above-mentioned discovery is that the scheme that combined clinical medication is infected as treatment drug resistance fungal provides thinking.

Ambroxol hydrochloride except alone there is anti-candida albicans planktonic cells and biomembranous activity except, inventor also chances on it under study for action can produce significantly collaborative antifungic action with fluconazol coupling. ABH and FLC use in conjunction has collaborative overriding resistance CA effect in vitro, and effect is obvious. The ABH of low dosage can make FLC that from 512 more than μ g/mL, the MIC value of drug resistance CA is down to 2 μ g/mL.

The present invention compared with prior art, has the following advantages and effect:

1. present invention demonstrates that, during ABH and FLC use in conjunction, it is possible to strengthen the FLC antibacterial activity to drug resistance CA, it is possible to produce collaborative antifungic action, and can the reversing drug resistance CA drug resistance to antifungal drug in triazole class, for the exploitation of new drug and old medicine newly with providing research direction. CA to FLC drug resistance, drug combination can make its minimum inhibitory concentration substantially reduce, and the FLC of ABH and the 2 μ g/mL of 128 μ g/mL share the fungus that can kill more than 80%, and concentration increases again, and effect is higher.

2.ABH application quantity clinically is big, safety, and it, to FLC antifungal potentiation, can expand its range of application, and reduce minimum effective Mlc of FLC, reducing the dosage of antifungal drug, thus reducing the generation of the untoward reaction of medicine, overcoming antifungal agent resistance problem clinically.

3. antifungal agent resistance is machine-processed and complicated, still has much mechanism to be still not very clear, if ABH is thorough to the antifungal enhanced sensitivity Mechanism Study of fluconazol, and must for overcoming antifungal agent resistance problem to provide new thinking.

Accompanying drawing explanation

Fig. 1. ambroxol hydrochloride and fluconazol coupling are to overriding resistance Candida albicans CA10 exercising result;

Fig. 2. ambroxol hydrochloride and fluconazol coupling are to overriding resistance Candida albicans CA16 exercising result;

Wherein: X-axis represents the concentration of fluconazol, Y-axis represents the concentration of ambroxol hydrochloride, what Z axis represented is the Δ E value under each drug regimen, collaborative or antagonism is represented respectively higher or lower than the value of plane (Δ E=0), value around plane represents unrelated effect, in the color encoding strip on figure right side, illustrate that the closer to upper end synergism is more strong.

Detailed description of the invention

Below in conjunction with accompanying drawing, the invention will be further described with embodiment.

Embodiment ambroxol hydrochloride measures with fluconazole antifungic action

1. material

1.1 medicines and reagent

Fluconazol (Fluconazole, FLC), Dalian Mei Lun Bioisystech Co., Ltd;

Ambroxol hydrochloride (Ambroxolhydrochloride, ABH), Dalian Mei Lun Bioisystech Co., Ltd;

Kerma (unit of kinetic energy) praises Candida chromogenic medium, Zhengzhou Bo Sai biological engineering company limited;

TTC-sand Borrow's culture medium, Qingdao high-tech park Hai Bo Bioisystech Co., Ltd;

Yeast extract, Beijing extensive and profound in meaning star biotechnology Co., Ltd;

Peptone, Beijing extensive and profound in meaning star biotechnology Co., Ltd;

Glucose, Chemical Reagent Co., Ltd., Sinopharm Group;

Agar powder, Beijing DingGuo ChangSheng Biology Technology Co., Ltd;

PBS phosphate buffer, Beijing DingGuo ChangSheng Biology Technology Co., Ltd;

Sodium hydroxide, state-run Shandong Dan County Organic Chemical Plant, lot number 940420;

Potassium dihydrogen phosphate, Shanghai is precious Fine Chemical Works newly, lot number 200602132.

RPMI1640 raw material medicated powder, GIBCO company of the U.S.;

3-(N-morpholino) propane sulfonic acid (MOPS), Beijing DingGuo ChangSheng Biology Technology Co., Ltd;

Menadione (Menadione), Sigma Co., USA;

XTT (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: take XTT powder 0.0500g, is dissolved in the autoclaved ringer's solution of 100mL and is made into the solution of 0.5mg/mL, filters with 0.22 μm of filter membrane and goes out; Adding the menadione acetone soln (take menadione 0.0860g and be dissolved in 5mL acetone) of the 10mmol/L of 10 μ L so that it is final concentration of 1 μm of ol/L, shake up, 4 DEG C keep in Dark Place.

Drug solution: fluconazol sterile distilled water dissolves, and is made into the storing solution of 2560 μ g/mL, filters subpackage; Ambroxol hydrochloride sterile distilled water dissolves, and is made into the storing solution of 5120 μ g/mL, filters subpackage. All medicinal liquids are in-20 DEG C of Refrigerator stores, standby.

PBS: weigh 12gPBS phosphate buffer to 1L volumetric flask, adds distilled water stirring and makes it be completely dissolved, then add aquae destillata to graduation mark, is dispensed into after reagent bottle at 121 DEG C autoclaving 30min, puts 4 DEG C of Refrigerator stores after cooling.

Yeast extract-peptone-glucose agar medium: glucose 10g, peptone 10g, yeast extract 5g, agar powder 10g, it is dissolved in water in 500mL conical flask, stirs rear 121 DEG C of sterilizing 30min, cool down rear 4 DEG C of Refrigerator stores standby.

RPMI1640 liquid medium: take RPMI1640 (containing L-glutaminate, without sodium bicarbonate) powder 2.08g, add 10% glucose solution 40ml (sugary final concentration 2%) and MOPS powder 6.906g, add distilled water to being about 200mL, pH is adjusted to be about 7.0 ± 0.1 at 22 DEG C by the NaOH solution of 1mol/L after mix homogeneously, before use with 0.22 μm of composite fibre membrane filtration sterilizing.

1.2 instruments

1.3 experimental strains

Quality-control strains: Candida albicans ATCC10231, pharmacology teaching and research room of Shandong University is so kind as to give;

Experimental strain: the Candida albicans that Qianfo Mount hospital clinical separates;

Identification of strains: experiment bacterial strain is cultivated 48 hours at the good Candida chromogenic medium 35 DEG C of Kerma (unit of kinetic energy), and bacterium colony is that green or emerald all bacterial strains are accredited as Candida albicans then through Shandong Center for Disease Control & Prevention's microbe research room with standard microbiology method.

Bacterium solution preparation: the Candida albicans thawed at room temperature preserved at-20 DEG C, is inoculated on TTC-sand Borrow's agar culture medium, cultivates 24h, takes well-developed single bacterium colony and again inoculate for 35 DEG C, cultivates 24h for 35 DEG C, to ensure that bacterial strain is in trophophase. Choosing some single relatively macrocolonies, PBS is configured to bacteria suspension, turbid with China's bacterial turbidity standard pipe ratio after vortice shaken well, adjusts sample cell consistent with standard pipe turbidity, and now the bacteria concentration of Candida albicans is about 1 × 10⁶CFU/mL, namely serial dilution obtains work bacterium solution, and carries out concentration checking with count plate.

2. content and method

2.1 ambroxol hydrochlorides measure with fluconazole overriding resistance Candida albicans effect

Chessboard method according to CLSIM27-A3 scheme, 4 times of working concentrations are become with RPMI-1640 fluid medium dilute liquid medicine, the concentration range of screening ABH and FLC use in conjunction, i.e. final concentration of 64～0.125 μ g/mL of final concentration of 256～4 μ g/mL, the FLC of ABH. FLC medicinal liquid 50 μ L is drawn respectively by concentration order from low to high, it is separately added into the 2nd～11 row of 96 hole flat boards, ABH medicinal liquid 50 μ L is drawn by concentration order from low to high, it is separately added into G～A row of each 96 hole flat boards, except arranging except the 12nd, each hole adds 100 μ L bacterium solution more respectively, and all the other hole RPMI-1640 culture fluid less than 200 μ L are supplied. Wherein H1 is growth control, and containing only bacterium solution not drug containing, the 12nd is classified as blank, containing only RPMI-1640 fluid medium. 96 hole flat boards of dosing are put after cultivating 24h in 35 DEG C of constant incubators by the requirement according to CLSIM27-A3 scheme, respectively with measuring OD with microplate reader after XTT load 2h and recording result. All experiments are in triplicate.

2.2 evaluation methodologys judge with result

2.2.1Loeweadditivity theoretical

Basic thought theoretical for Loeweadditivity (LA) thinks that medicine can not interact with itself, and the concentration (equivalence site) that therefore alone for medicine or coupling produce identical drug effect compares. It analyzes method mark Mlc index method (fractionalinhibitoryconcentrationindex, FICI), is expressed as follows:

Σ FIC=FIC_A+FIC_B=C_A/MIC_A+C_B/MIC_B

MIC_AAnd MIC_BBe respectively medicine A and B alone time minimal inhibitory concentration, C_AWith C_BRespective concentration during identical drug effect is reached when being two medicine couplings. FICI > 4 is antagonism, and FICI is for being added or unrelated effect between 0.5 with 4, and FICI≤0.5 is defined as synergism.

2.2.2Blissindependence theoretical

Blissindependence (BI) theory is with Δ E (under each concentration of medicine, fungus grows the theoretical value of percentage rate and the difference of experiment value) for Data Analysis Model. Δ E model formulation is as follows:

Δ E=E_A×E_B-E_comb

Wherein, E_AFor the rate of growth of A prescription used time fungus, E_BFor the rate of growth of B prescription used time fungus, E_combThe rate of growth of fungus during for A medicine and B medicine coupling. During interpretation of result, by Σ SYN and Σ ANT, the i.e. interaction adding and judging for index two kinds of medicines of all positive value delta E and negative value Δ E in 96 orifice plates. When the absolute value of Δ E < when 100%, is expressed as weak collaborative or weak antagonism; When the absolute value of Δ E is between 100% and 200%, it is expressed as medium collaborative or antagonism, when the absolute value of Δ E is more than 200%, is expressed as collaborative by force or antagonism.

3. result

3.1 ambroxol hydrochlorides and fluconazole overriding resistance Candida albicans exercising result

3.1.1 minimum effective Mlc of ambroxol hydrochloride and fluconazole

In each hole, the computational methods of fungus growth percent are:

Fungus growth percent=(each hole OD value-blank control wells OD value)/growth control hole OD value

Calculating each hole fungus growth percent in flat board by above-mentioned formula, taking the minimum coupling drug level that can suppress fungus growth 80% is interpretation terminal.

When ABH and FLC combines antifungic action, drug resistance Candida albicans is presented strong synergism, but for the sensitive strain of Candida albicans and non-white candidiasis without synergism. Now persister CA10 and CA16 growth percent experimental result in dosing 96 orifice plate is listed as follows shown in (table 1-3).

Table .1. chessboard represents that the drug combination overriding resistance Candida albicans CA10 of ABH and FLC grows percentage rate (marking with the medicine the best use of combination Lycoperdon polymorphum Vitt of FICI method conversion).

Table .2. chessboard represents that the drug combination overriding resistance Candida albicans CA16 of ABH and FLC grows percentage rate (marking with the medicine the best use of combination Lycoperdon polymorphum Vitt of FICI method conversion).

3.1.2FICI the synergism of model and Δ E model evaluation ABH and FLC coupling

The evaluation index of FICI model is FICI value, and FICI≤0.5 item is defined as synergism. As can be seen from Table 3, the FICI value of each combination is respectively less than 0.5, shows as stronger synergism.

The evaluation index of Δ E model is Σ SYN and Σ ANT, and as can be seen from Table 3, each summation organizing ∑ SYN and ∑ ANT is all far longer than 200%, shows as strong synergism, graphics such as Fig. 1 and Fig. 2 of ABH and FLC coupling antagonism CA10 and CA16. Can find out that from figure different pharmaceutical combination is respectively provided with strong synergism intuitively. Picture and above-mentioned numerical analysis have concordance.

Table .3. is with FICI model and Δ E model evaluation ambroxol hydrochloride and fluconazole medication antifungic action

Explain: FLC: fluconazol; ABH: ambroxol hydrochloride; MIC: minimum inhibitory concentration; MIC_A: the minimum inhibitory concentration of fluconazol when medicine is alone; C_A: the minimum inhibitory concentration of fluconazol during drug combination; MIC_B: the minimum inhibitory concentration of ambroxol hydrochloride when medicine is alone; C_B: the minimum inhibitory concentration of ambroxol hydrochloride during drug combination; FICI: mark Mlc index; All positive value delta E sums in Σ SYN:96 orifice plate; All negative value Δ E sums in Σ ANT:96 orifice plate.

The specific embodiment of the present invention is described in conjunction with accompanying drawing although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme, those skilled in the art need not pay various amendments or deformation that creative work can make still within protection scope of the present invention.

Claims

1. an ambroxol hydrochloride associating fluconazol application in preparing antifungal products.

2. apply as claimed in claim 1, it is characterized in that: described product is medicine.

3. apply as claimed in claim 1, it is characterized in that: described fungus is Candida albicans.

4. apply as claimed in claim 1, it is characterized in that: valid density proportioning when described ambroxol hydrochloride and fluconazole application: fluconazol: ambroxol hydrochloride=1:64.

5. apply as claimed in claim 1, it is characterized in that: during described application, the minimum inhibitory concentration of ambroxol hydrochloride is: 128 μ g/mL.

6. apply as claimed in claim 1, it is characterized in that: during described application, the minimum inhibitory concentration of fluconazol is: 2 μ g/mL.

7. an antifungal products, is characterized in that: with ambroxol hydrochloride and fluconazol for main active.

8. antifungal products as claimed in claim 7, is characterized in that, the valid density proportioning of described ambroxol hydrochloride and fluconazol is fluconazol: ambroxol hydrochloride=1:64.

9. antifungal products as claimed in claim 7, is characterized in that, the least concentration of described ambroxol hydrochloride is: 128 μ g/mL.

10. antifungal products as claimed in claim 7, is characterized in that, the least concentration of described fluconazol is: 2 μ g/mL.