Application of echinocandins drug combined with calcineurin inhibitor in preparation of antifungal product

Abstract

The invention provides an application of an echinocandins drug combined with a calcineurin inhibitor in preparation of an antifungal product. Research finds that compared with a wild type strain, after an echinocandin drug caspofungin with the concentration lower than MIC is added into high-density calcineurin knockout candida glabrata, a quorum sensing phenomenon can be observed, that is, growth is inhibited. Besides, by further combined use of the calcium phosphatase inhibitor and caspofungin, a similar inhibition phenomenon is also observed, that is, the growth of high-density bacteria is significantly inhibited, which indicates that the combined strategy has potential application value in resisting Candida glabrata infection. Therefore, the invention also provides a composition comprising the echinocandin medicine and the calmodulin inhibitor, the composition is used for inhibiting the growth of the candida glabrata, and the composition is particularly suitable for high-density candida glabrata and has a remarkable antibacterial effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the use of echinocandin drugs combined with calcineurin inhibitors in the preparation of antifungal products, including a composition of echinocandin drugs and calcineurin inhibitors, and a method for inhibiting the growth of Candida glabrata by synergistic action of echinocandin drugs and calcineurin deficiency. Background Art

[0002] Non-Candida albicans species are a class of fungi different from Candida albicans, mainly including Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, etc. Unlike Candida albicans, non-Candida albicans generally do not produce hyphae, but mainly exist in the form of biofilms, which provide them with a stable extracellular matrix structure, thereby helping microbial cells resist various harsh environmental pressures, such as ultraviolet radiation, osmotic pressure stress, high temperature, antibiotics, immune attacks, etc. Therefore, the treatment of biofilms has become a very difficult problem and is the main cause of microbial infections.

[0003] Among them, Candida glabrata is a common conditional pathogen that is widely found in the skin, oral cavity, intestinal tract and reproductive tract of humans and animals. Under the influence of factors such as low immune function or the use of broad-spectrum antibiotics, Candida glabrata is prone to cause infection, especially in hospital-acquired infections. Due to its inherent or acquired resistance to multiple antifungal drugs, the treatment of Candida glabrata infection faces great challenges.

[0004] At present, caspofungin, as an echinocandin antifungal drug, has been widely used in the clinical treatment of various fungal infections, including Candida glabrata. However, although caspofungin has a significant effect in inhibiting fungal cell wall synthesis, its effect in high-concentration strains is not significant, and Candida glabrata's resistance to caspofungin is gradually increasing. Therefore, it is necessary to provide an anti-non-albicans Candida albicans product with better inhibitory effect. Summary of the invention

[0005] In order to solve the problem in the prior art that caspofungin has a poor inhibitory effect on non-albicans Candida albicans, especially in high-density Candida glabrata strains, the present invention provides a method for inhibiting the growth of high-density Candida glabrata by synergistic action of echinocandins and calcineurin deficiency, and based on this, echinocandins are combined with calcineurin inhibitors for use in the preparation of antifungal products, including a composition of echinocandins and calcineurin inhibitors that synergistically act on Candida glabrata to inhibit its growth, thereby combating Candida glabrata infection.

[0006] Calcineurin plays an important role in fungal growth and drug resistance. It is a calcium-dependent serine / threonine phosphatase that affects fungal survival and reproduction by regulating a variety of cellular physiological processes, including cell cycle, stress response and drug resistance. Inhibiting the activity of calcineurin has been shown to enhance the effect of antifungal drugs. For example, the mechanism of action of the calcineurin inhibitor tacrolimus (FKS506) is mainly achieved by inhibiting the activity of calcineurin. Calcineurin is responsible for the activation of the gene transcription factor NFAT (nuclear factor activated T cells) required for T cell activation, and tacrolimus binds to the cytoplasmic protein FKBP12 to form a complex, thereby inhibiting the activity of calcineurin, preventing the activation and proliferation of T cells, thereby achieving the effect of suppressing the immune response.

[0007] The present invention has found through research that compared with the wild-type strain, the high-density glabrata Candida albicans (OD 600 0.0001~0.1) after adding the echinocandin drug caspofungin at a concentration lower than the MIC (0.6μg / mL), a quorum sensing phenomenon can be observed. Specifically, wild-type and calcineurin gene knockout bacteria were treated with caspofungin at a concentration lower than the MIC, and the growth of high-density calcineurin knockout bacteria was significantly inhibited after caspofungin treatment. Although this inhibition will be overcome with the further growth of high-concentration bacteria, the growth of low-density colonies is always consistent with that of wild-type bacteria. This discovery provides us with a new idea, combining echinocandin drugs with substances that inhibit calcineurin to synergistically act on Candida glabrata to inhibit its growth and to combat Candida glabrata infection. Therefore, in the implementation of the present invention, the calcineurin inhibitor tacrolimus and caspofungin are further used in combination, and the simple knockout bacteria are further externalized into inhibitory drugs, and an inhibitory effect on high-density Candida glabrata is also observed. Therefore, the present invention also provides a composition comprising an echinocandin drug and a calcineurin inhibitor for inhibiting the growth of Candida glabrata. The composition is particularly suitable for high-density Candida glabrata and has a significant antibacterial effect.

[0008] The technical solution adopted by the present invention is: the use of an echinocandin drug combined with a calcineurin inhibitor in the preparation of an antifungal product, characterized in that the fungus is non-albicans Candida albicans, and the optical density OD of the fungus is 600 Not less than 0.0001.

[0009] Specifically, the non-albicans Candida include Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei, preferably Candida glabrata.

[0010] Specifically, the optical density OD of Candida glabrata 600It is 0.0001~0.1.

[0011] Specifically, the echinocandin drug is selected from one or more of caspofungin, micafungin, and anidulafungin, preferably caspofungin.

[0012] Specifically, the calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus, and cyclosporine, preferably tacrolimus.

[0013] Specifically, the application includes: application of the echinocandin drug caspofungin combined with the calcineurin inhibitor tacrolimus in the preparation of an anti-Candida glabrata product; wherein the amount of caspofungin used is preferably not less than 0.1 μg / mL, the amount of tacrolimus used is preferably not less than 1 mg / mL, and the optical density OD of Candida glabrata is 600 It is preferably 0.0001 to 0.1 (more preferably 0.001 to 0.1).

[0014] The present invention also provides a composition, including an echinocandin drug and a calcineurin inhibitor; the echinocandin drug is selected from one or more of caspofungin, micafungin, and anidulafungin; the calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus, and cyclosporin. The composition is used to prepare an antifungal product, the fungus is non-albicans Candida albicans, and the optical density OD of the fungus is 600 Not less than 0.0001.

[0015] The present invention also provides a method for inhibiting the growth of Candida glabrata by synergistic action of echinocandin drugs and calcineurin deficiency, comprising: treating Candida glabrata with calcineurin inhibition and applying echinocandin drugs; wherein the optical density OD of the Candida glabrata is 600 Not less than 0.0001

[0016] Preferably, the echinocandin drugs are selected from one or more of caspofungin, micafungin and anidulafungin.

[0017] Preferably, the calcineurin inhibition treatment comprises: knocking out the calcineurin encoding gene in the Candida glabrata genome, and / or applying a calcineurin inhibitor to the Candida glabrata.

[0018] Preferably, after knocking out the calcineurin encoding gene in the Candida glabrata genome, the applied concentration of the echinocandin drug caspofungin is not less than 0.3 μg / mL.

[0019] Preferably, the calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus and cyclosporine.

[0020] Beneficial effects of the present invention:

[0021] The present invention combines an echinocandin drug (such as caspofungin) with a calcineurin inhibitor (such as tacrolimus) to inhibit the activity of calcineurin, thereby enhancing the effect of the echinocandin drug and effectively improving the inhibitory effect on high-density Candida glabrata, thereby solving the problem in the prior art that caspofungin alone has a poor inhibitory effect on non-albicans Candida albicans (especially high-density Candida glabrata). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are spot test diagrams under the induction of caspofungin in Examples 1 and 2 of the present invention, wherein the first and second rows compare the growth of the wild-type and knockout bacteria; the first and third rows compare the effect of tacrolimus on the growth of the wild-type strain.

[0023] Figure 2 This is the MIC graph of Candida glabrata to caspofungin in 96-well plate culture in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0025] Example 1: Spot experiment of caspofungin-induced calcineurin gene knockout bacteria

[0026] The wild-type Candida glabrata strain and the Candida glabrata strain with phosphatase gene knockout were respectively prepared (the preparation process refers to Miyazaki T, Yamauchi S, Inamine T, Nagayoshi Y, Saijo T, Izumikawa K, Seki M, Kakeya H, Yamamoto Y, Yanagihara K, Miyazaki Y, Kohno S. Roles of calcineurin and Crz1 in antifungal susceptibility and virulence of Candida glabrata. Antimicrob Agents Chemother. 2010Apr; 54(4): 1639-43. doi: 10.1128 / AAC.01364-09. Epub 2010Jan 25.PMID:20100876;PMCID:PMC2849377.) was activated twice on a YPD plate, i.e., streaked on a YPD plate and cultured overnight in a 37°C incubator to ensure its activity. Caspofungin (CASP) was prepared to a mother solution concentration of 48 μg / mL, and after the sterilized YPD agar medium was cooled to a suitable temperature, different volumes of drug solution were added to prepare YPD plate medium with caspofungin concentrations of 0.10 μg / mL and 0.12 μg / mL, respectively, and poured into a plate solid medium of similar thickness to solidify it.

[0027] Dilute the bacterial solution in the test tube with sterile water to OD 600 =1, and then the diluted bacterial solution was serially diluted 10 times on a microplate, with a total of four dilutions (OD 600 10 respectively -4 , 10 -3 , 10 -2 , 10 -1 ,), take 5 μL of each and drop them neatly on the YPD plate containing resistance. Try to ensure that the four groups can be on the same horizontal line for easy observation. Without adding any drugs, the same spot test was performed on the YPD plate as a control. The plate was cultured in a 37℃ incubator for 24 hours to observe the phenomenon ( Figure 1 ).

[0028] The results of the spot experiment showed that the calcineurin knockout bacteria observed a quorum sensing phenomenon after adding caspofungin at a concentration lower than the MIC, that is, the growth of high-density inoculated colonies was inhibited, while the growth of low-density colonies was consistent with that of wild-type bacteria. Specifically, at a caspofungin concentration of 0.1 μg / mL, it can be seen that the bacterial cell density was 10 -3When the concentration of calcineurin knockout bacteria was too low to degrade or overcome caspofungin and the quorum sensing effect caused the bacteria to not grow normally, the calcineurin knockout bacteria showed obvious quorum sensing, while the wild-type bacteria did not. That is, when the density of calcineurin knockout bacteria was lower, the growth condition was better than when the quorum sensing phenomenon occurred, indicating that the quorum sensing phenomenon requires a certain accumulation of cells to occur. At the same time, it was found that after a period of cultivation, high-density bacteria had a tendency to degrade and overcome caspofungin, and the bacteria resumed growth.

[0029] Example 2: Spot test of caspofungin combined with the calcineurin inhibitor tacrolimus

[0030] This example describes the antibacterial experimental method and effect verification of the combined use of caspofungin and tacrolimus. First, caspofungin below the minimum inhibitory concentration (MIC) was used in combination with tacrolimus at a concentration of 1 mg / mL. The specific steps are as follows:

[0031] Different concentrations of caspofungin were added to the YPD plate medium, and then 100 μL of 1 mg / mL tacrolimus solution was evenly spread on the surface of the plate. The solution was allowed to dry naturally and then used. The strain that had been cultured to the logarithmic growth phase was suspended in sterile water and diluted to an optical density of OD 600 = 1. The obtained bacterial solution was diluted 10-fold in series on a 96-well microplate, and four groups of gradient dilutions were performed. 5 μL of each dilution was dripped on the YPD plate containing different concentrations of caspofungin and tacrolimus in sequence, ensuring that each drop was in the same straight line for easy comparison. The culture plate was cultured under appropriate conditions and the growth of the colonies was observed.

[0032] The results show that ( Figure 1 ), the antibacterial effect of caspofungin combined with tacrolimus was significantly better than that of caspofungin alone, reflecting the synergistic antibacterial effect of the combined use.

[0033] Example 3: MIC experiment in 96-well plate

[0034] In order to further study the changes in the tolerance of calcineurin knockout bacteria to caspofungin, the MIC of caspofungin was determined in 96-well plate culture of Candida glabrata.

[0035] (1) Preparation of bacterial suspension: After the strain was activated twice on the YPD plate, 5 colonies with a diameter of >1 mm were picked from the YPD plate, resuspended in sterile water, and oscillated in an oscillator for 15 seconds to prepare a bacterial suspension. The concentration of the bacterial suspension was adjusted using a hemocytometer and YPD liquid culture medium to a concentration of 5×10 6CFU / mL is approximately equal to 0.5 McFarland turbidity. Dilute the prepared bacterial suspension 1000 times with YPD liquid medium to make the concentration between 1×10 3 ~5×10 3 CFU / mL range.

[0036] (2) Preparation of drug sensitivity plate: For the blank control, 200 μL of YPD liquid medium was added to well 1 of each row of the 96-well plate. 80 μL of YPD liquid medium and 20 μL of drug solution were added to wells 2 to 12 respectively. 20 μL of sterile water was added to well 12 as a positive control. The drugs in wells 2 to 11 were diluted 10-fold so that the final drug concentrations in each well were 4.8, 2.4, 1.2, 0.6, 0.3, 0.15, 0.08, 0.03, 0.02, and 0.01 μg / mL, respectively.

[0037] (3) Inoculation and culture: Use a multichannel pipette to add 80 μL of the prepared bacterial solution to wells 3 to 12 of a 96-well plate to a final inoculation concentration of 0.5 × 10 3 ~2.5×10 3 CFU / mL. Each strain was tested in triplicate and the results were observed after 72 hours of static culture at 30°C in an incubator.

[0038] (4) Reading of MIC value: Visual observation was used. The MIC value of the screening compound was determined as the minimum drug concentration when the growth of the growth control was reduced by more than 50%. The MIC value of caspofungin was determined as the minimum drug concentration when the growth of the control was reduced by more than 50%.

[0039] The results are as follows Figure 2 As shown, it can be seen that the MIC of caspofungin against Candida glabrata is 0.3μg / mL for the defective cnb1Δ bacteria and 0.6μg / mL for the wild type. It was found that the calcineurin knockout bacteria had a worse tolerance to caspofungin than the wild type. It is inferred that the knockout of calcineurin may change the cell wall components of the bacteria, resulting in a decrease in its tolerance.

[0040] In summary, calcineurin knockout bacteria have increased sensitivity to caspofungin, and this phenomenon was specifically combined with the inhibitory drug tacrolimus to further reveal a stronger antibacterial effect. These findings provide a scientific basis for the development of new antifungal methods.

[0041] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. Use of an echinocandin drug combined with a calcineurin inhibitor in the preparation of an antifungal product, characterized in that: The fungus is non-albicans Candida albicans, and the optical density OD of the fungus 600 Not less than 0.0001.

2. The use according to claim 1, characterized in that The non-albicans Candida include Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei.

3. The use according to claim 1, characterized in that The echinocandin drugs are selected from one or more of caspofungin, micafungin and anidulafungin.

4. The use according to claim 1, characterized in that The calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus and cyclosporine.

5. The use according to claim 1, characterized in that The echinocandin drug is caspofungin, the calcineurin inhibitor is tacrolimus, and the fungus is Candida glabrata.

6. The use according to claim 5, characterized in that When preparing anti-Candida glabrata products, the amount of caspofungin used is not less than 0.1 μg / mL, the amount of tacrolimus used is not less than 1 mg / mL, and the optical density OD of Candida glabrata is 600 It is 0.0001~0.

1.

7. A composition, characterized in that These include echinocandins and calcineurin inhibitors; The echinocandin drugs are selected from one or more of caspofungin, micafungin, and anidulafungin; The calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus and cyclosporine.

8. A method for inhibiting the growth of Candida glabrata by synergistic action of echinocandins and calcineurin deficiency, characterized in that: include: Calcineurin inhibition treatment is adopted for Candida glabrata, and echinocandin drugs are applied; wherein the optical density OD of the Candida glabrata is 600 Not less than 0.0001.

9. The method according to claim 8, characterized in that The echinocandin drugs are selected from one or more of caspofungin, micafungin, and anidulafungin; The calcineurin inhibition treatment includes: knocking out the calcineurin encoding gene in the Candida glabrata genome, and / or applying a calcineurin inhibitor to the Candida glabrata.

10. The method according to claim 9, characterized in that After knocking out the calcineurin encoding gene in the Candida glabrata genome, the applied concentration of the echinocandin drug caspofungin was no less than 0.3 μg / mL; The calcineurin inhibitor is selected from one or more of pimecrolimus, tacrolimus and cyclosporine.

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