Application of Apo-E mimetic peptide COG1410 in the preparation of drugs for inhibiting mycobacteria

Abstract

The present invention provides the use of the Apo-E mimetic peptide COG1410 in the preparation of a drug for inhibiting mycobacteria, belonging to the technical field of biomedicine. The present invention discovers through research that COG1410 can rapidly and effectively inhibit mycobacteria, especially Mycobacterium abscessus, by disrupting the bacterial cell membrane, and at the same time has anti-inflammatory activity. COG1410 of the present invention has good stability in plasma, a low frequency of inducing resistance in Mycobacterium abscessus, and can also be used in combination with a variety of commonly used antibiotics to achieve the purpose of highly inhibiting Mycobacterium abscessus, providing a new option for clinically combating drug-resistant Mycobacterium abscessus and treating MAB lung disease drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of Apo-E mimetic peptide COG1410 in the preparation of drugs for inhibiting mycobacteria. Background Art

[0002] Nontuberculous Mycobacterium (NTM) refers to mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae. Although NTM widely exists in the environment, most of them can cause diseases in humans or animals. In particular, they can cause infections in organs such as the lungs, lymph nodes, joints, and central nervous system, or even systemic disseminated infections in susceptible individuals, among which the lungs are most easily infected and develop into NTM lung disease. In recent years, the incidence and prevalence of NTM lung disease have increased rapidly globally, and the incidence in many countries even exceeds that of pulmonary tuberculosis. Since the clinical manifestations and chest imaging manifestations of NTM lung disease are very similar to those of pulmonary tuberculosis, acid-fast bacilli can be found in sputum, and it is often difficult to distinguish NTM lung disease from pulmonary tuberculosis, which brings new challenges to the prevention and control of tuberculosis.

[0003] Mycobacterium abscessum complex (MABc) is one of the main NTM species that cause NTM lung disease, skin lesions, disseminated lesions, etc., and its incidence has been increasing year by year. Guidelines at home and abroad recommend a regimen based on macrolides combined with multiple antibiotics to treat MAB lung disease until the patient's sputum culture turns negative for more than one year, and then adjust the regimen according to further drug sensitivity results and clinical observations. For patients with failed drug treatment, repeated infections, antibiotic resistance, or intolerance to antibiotic treatment, and with relatively limited pulmonary lesions, surgical assistance combined with antibiotic treatment can be considered, but there may be complications such as postoperative bronchopleural fistula. The treatment of MAB is relatively time-consuming, difficult, costly, and ineffective compared to other NTM.

[0004] In addition, due to the lipid-rich cell wall of MAB, its cell wall has strong hydrophobicity, which is not conducive to the entry of water-soluble substances and becomes an obstacle to effective chemotherapy. The "Diagnosis and Treatment Guidelines for Nontuberculous Mycobacteriosis" jointly developed by the Chinese Medical Association's Tuberculosis Branch in 2020 clearly states that so far, there is no drug that can truly treat MAB infections. The 17 anti-MAB drugs mentioned in the current guidelines are all drugs used to treat other diseases, including anti-tuberculosis drugs (such as isoniazid, rifampicin, ethambutol, etc.), macrolides (such as azithromycin, clarithromycin), and quinolones (such as moxifloxacin). However, MAB is resistant to all conventional first-line anti-tuberculosis drugs. Macrolides, once regarded as the most effective and basic anti-NTM drugs, have been used for nearly 20 years, and some MAB strains have developed resistance or high resistance to them. Multidrug-resistant or even pan-drug-resistant MAB has put clinicians in a serious situation where there are no drugs available.

[0005] In summary, even if multiple antibiotics with long treatment courses and high costs are used in clinical treatment, due to the high drug resistance of MAB, MAB lung disease is difficult to treat, has poor efficacy, and is prone to recurrence, making it another major challenge to global public health security after Mycobacterium tuberculosis. Therefore, it is urgent to search for and develop new antibacterial drugs against MAB.

[0006] The research of Cogentus Pharmaceuticals, Inc. in the United States (CN200580038189.2) found that the receptor-binding fragment (133-149aa) of Apo-E (also known as COG133) can mimic the main biological activities of the whole Apo-E protein, including anti-inflammatory activity, anti-excitatory neurotoxicity, and neuroprotection and neuroregeneration. After further optimization and modification, the sequence was reduced to 138-149, and the amino acids at positions 140 and 145 were replaced with amino isobutyric acid to obtain the second-generation mimetic peptide CST1410 (also known as COG1410). However, the bactericidal activity of this mimetic peptide, especially against Mycobacterium abscessus, has not been disclosed in the prior art. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide the application of Apo-E mimetic peptide COG1410 in the preparation of drugs for inhibiting mycobacteria. COG1410 has good bactericidal activity against mycobacteria, especially multidrug-resistant Mycobacterium abscessus, and can effectively treat diseases caused by MAB infection.

[0008] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0009] The application of Apo-E mimetic peptide COG1410 in the preparation of drugs for inhibiting mycobacteria.

[0010] Preferably, the mycobacterium includes one or more of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium abscessus.

[0011] Preferably, the Mycobacterium abscessus is a multi-drug resistant Mycobacterium abscessus.

[0012] Preferably, the Apo-E mimetic peptide COG1410 disrupts the integrity of the mycobacterial cell membrane.

[0013] The present invention also provides an application of the Apo-E mimetic peptide COG1410 in the preparation of a drug for treating MAB lung disease.

[0014] The present invention also provides an application of the combined use of the Apo-E mimetic peptide COG1410 and an antibiotic in the preparation of a drug for inhibiting mycobacteria.

[0015] Preferably, the antibiotic includes one or more of rifampicin, kanamycin, ethambutol, amikacin, ciprofloxacin, and clarithromycin.

[0016] Preferably, the concentration of the Apo-E mimetic peptide COG1410 is 2 - 20 μg / mL, and the concentration of the antibiotic is 0.2 - 20 μg / mL.

[0017] Preferably, the drug further includes a pharmaceutically acceptable excipient.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Designing a novel antibacterial peptide based on the human-derived apolipoprotein E - ApoE belongs to a brand-new antibacterial drug design concept. COG1410 is a very promising new drug for neuroprotection and has very strong anti-inflammatory activity. The present invention comprehensively evaluates the bactericidal activity and clinical application prospects of COG1410 against mycobacteria from three aspects: molecular mechanism, cellular level, and animal model. The research shows that COG1410 can quickly and effectively inhibit mycobacteria, especially Mycobacterium abscessus, by disrupting the bacterial cell membrane, and at the same time has anti-inflammatory activity. The COG1410 of the present invention has good stability in plasma, a low frequency of inducing resistance in Mycobacterium abscessus, and can be combined with a variety of commonly used antibiotics to achieve the purpose of highly inhibiting Mycobacterium abscessus, providing a new option for clinically combating drug-resistant Mycobacterium abscessus and treating drugs for MAB lung disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : In vitro bactericidal kinetics experiment of the Apo-E mimetic peptide COG1410;

[0021] Figure 2 : Anti-inflammatory activity of the Apo-E mimetic peptide COG1410;

[0022] Figure 3 : Transmission electron micrographs of the integrity of the cell membrane of Mycobacterium abscessus in the COG1410 treatment group and the negative control group;

[0023] Figure 4 : Effects of the COG1410 treatment group and the ethambutol treatment group on the leakage of intracellular ATP content of Mycobacterium abscessus;

[0024] Figure 5 : Results of the frequencies of emergence of resistance in Mycobacterium abscessus induced by the COG1410 treatment group and the rifampicin treatment group;

[0025] Figure 6 : Results of the bactericidal activities of the COG1410 treatment group and the control group in 50% plasma;

[0026] Figure 7 : Effects of the COG1410 treatment group and the negative control group on the activity of intracellular Mycobacterium abscessus.

[0027] Among them, CST1410 in the drawings is COG1410. Detailed implementation manners

[0028] Based on the Apo-E mimetic peptide COG1410, the present invention studies its bactericidal activity and corresponding antibacterial mechanism, and provides the application of the Apo-E mimetic peptide COG1410 in the preparation of drugs for inhibiting mycobacteria, which can produce good bactericidal activity against mycobacteria. The mycobacteria of the present invention preferably include one or more of Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium abscessus. Among them, the Mycobacterium abscessus is preferably a multi-drug resistant Mycobacterium abscessus; the Mycobacterium abscessus is such as Mycobacterium abscessus ATCC19977, the Mycobacterium tuberculosis is such as Mycobacterium tuberculosis H37Rv, and the Mycobacterium smegmatis is such as Mycobacterium smegmatis Mc 2 155. The minimum bactericidal concentrations of the Apo-E mimetic peptide COG1410 of the present invention against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis Mc 2 155, Mycobacterium abscessus ATCC19977 and Mycobacterium abscessus MAB-hun12 are 16 μg / mL, 16 μg / mL, 8 μg / mL and 8 μg / mL respectively. It is worth noting that the breakpoint for clinically judging the resistance of Mycobacterium abscessus to clarithromycin is 16 μg / mL. Therefore, the MIC of COG1410 against mycobacteria is ≤16 μg / mL, especially the MIC of COG1410 against Mycobacterium abscessus is <16 μg / mL. This shows that clinically, COG1410 has great potential to be used as an anti-drug resistant Mycobacterium abscessus and a drug for treating MAB lung disease.

[0029] In the present invention, COG1410 rapidly and effectively kills mycobacteria by disrupting the bacterial cell membrane.

[0030] The present invention further provides the use of the Apo-E mimetic peptide COG1410 in the preparation of a medicament for treating MAB lung disease, so as to provide guidance for the treatment of MAB lung disease and offer a new option for its medication. The MAB lung disease described in the present invention refers to NTM lung disease caused by infection with Mycobacterium abscessus complex (MABs) as the pathogenic bacterium.

[0031] The present invention also provides the use of the combined use of the Apo-E mimetic peptide COG1410 and an antibiotic in the preparation of a medicament for inhibiting mycobacteria. In the present invention, the antibiotic preferably includes one or more of rifampicin, kanamycin, ethambutol, amikacin, ciprofloxacin, and clarithromycin. The concentration of the Apo-E mimetic peptide COG1410 is preferably 2-20 μg / mL, more preferably 4-16 μg / mL, and the concentration of the antibiotic is 0.2-20 μg / mL, more preferably 0.5-16 μg / mL.

[0032] In the present invention, the medicament further includes pharmaceutically acceptable excipients, and the excipients include one or more of binders, excipients, diluents, disintegrants, antioxidants, dispersants, wetting agents, solubilizers, buffers, and surfactants. The medicament described in the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously, or nasally.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] In vitro bactericidal activity of the Apo-E mimetic peptide COG1410

[0036] A multi-drug resistant Mycobacterium abscessus strain MAB was isolated and collected from a clinical sample.

[0037] "Synchronization" of bacterial culture: The bacterial strain frozen in an -80°C refrigerator was streaked in a "four-zone" manner on a 7H10 agar medium and cultured in a 37°C incubator for 48 h. A single colony was picked and inoculated into 4 mL of 7H9 liquid medium (add the corresponding antibiotic if necessary), and cultured with shaking on a 37°C shaker until OD 600 was 0.5-1.0. The bacterial cells were collected by centrifugation, washed twice with fresh 7H9 medium, and resuspended in fresh 7H9 medium, and OD 600 was measured for standby.

[0038] Design the Apo-E mimetic peptide COG1410 to conduct bactericidal activity tests on four mycobacteria, namely Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis Mc 2 155, the standard strain Mycobacterium abscessus ATCC19977, and the multidrug-resistant Mycobacterium abscessus strain MAB-hun12.

[0039] Table 1 Minimum inhibitory concentration of COG1410 against several mycobacteria

[0040]

[0041] The results are shown in Table 1: The minimum inhibitory concentration of COG1410 against Mycobacterium abscessus is 8 μg / mL, 5.5 μM. There is no difference in sensitivity between the standard strain (ATCC19977) and the clinical isolate MAB of Mycobacterium abscessus. Considering that there has been no new drug for the treatment of MAB infection, the MIC of COG1410 is less than 16 μg / mL, indicating that COG1410 has the potential to be applied clinically.

[0042] Further in vitro bactericidal kinetics experiments were carried out, and the results are as Figure 1 shown. In PBS, COG1410 (1×MIC) can reduce the number of MAB bacteria from 10 8 to 10 5 within 5 minutes, which means that 99% of the bacteria are killed; if the concentration of COG1410 is increased to 40 μg / mL (5×MIC), COG1410 can almost completely kill the MAB cells within 5 minutes.

[0043] Example 2

[0044] Anti-inflammatory activity of the Apo-E mimetic peptide COG1410

[0045] Prepare different doses of COG1410, 0.7 mg / kg and 2.8 mg / kg, and inject them into C57BL / 6J mice via intravenous injection. Then, an inflammatory model of mice is constructed by injecting LPS via the tail vein. At the same time, the mimetic peptide COG125 (ineffective polypeptide) from Apo-E is used as a negative control, and COG133 (which has been proven to have anti-inflammatory efficacy, CN200580038189.2) is used as a positive control.

[0046] The results are as Figure 2 shown. Compared with the control group, COG1410 can reduce the concentration of the inflammatory factor - tumor necrosis factor TNFα in the plasma by about 70%. The above results indicate that COG1410 not only has antibacterial activity but also has anti-inflammatory activity, and can exert its effect through intravenous injection.

[0047] Example 3

[0048] Observation of the effect of COG1410 treatment on the cell membrane integrity of abscesses by transmission electron microscopy

[0049] "Synchronization" of bacterial culture: The bacterial strain stored in an -80 °C refrigerator was streaked in a "four-zone" manner on 7H10 agar medium and cultured in a 37 °C incubator for 48 h. A single colony was picked and inoculated into 4 mL of 7H9 liquid medium (add corresponding antibiotics when needed), and cultured with shaking on a 37 °C shaker until OD 600 reached 0.5 - 1.0. The bacterial cells were collected by centrifugation, washed twice with fresh 7H9 medium, and resuspended in fresh 7H9 medium, and OD 600 was measured for standby.

[0050] The synchronized drug-resistant Mycobacterium abscessus MAB was inoculated into 7H9 and cultured with shaking at 37 °C until the logarithmic growth phase. The bacterial cells were collected by centrifugation, washed once with 1×PBS, and then resuspended in PBS, and OD 600 was adjusted to 0.5. 1×MIC COG1410 was added and treated at 37 °C for 2 h. The untreated cells were used as negative controls, and the negative control group was added with the same volume of PBS as the COG1410 treatment group. The bacterial cells were collected by centrifugation, washed twice with PBS, and then added with glutaraldehyde fixative for electron microscopy, fixed in a 4 °C refrigerator for 2 h, and then sent to the testing center of Nanjing University of Chinese Medicine for transmission electron microscopy observation.

[0051] It was shown by Figure 3 the results that the cell membranes of the untreated bacterial cells were intact, while the cell membranes of the bacterial cells treated with COG1410 were damaged and the contents leaked.

[0052] Example 4

[0053] Whether COG1410 causes perforation of Mycobacterium abscessus strains

[0054] The drug-resistant Mycobacterium abscessus MAB was synchronized by the method of "synchronization" of bacterial culture described in Example 3, and the synchronized drug-resistant Mycobacterium abscessus MAB was cultured to the logarithmic growth phase. For each reaction, 5 mL of bacterial cells were collected by centrifugation, washed once with 50 mM PBS (pH 7.0), and then resuspended in 500 μL of 50 mM HEPES (pH 7.0) and 0.2% glucose buffer solution to concentrate 10 times to increase the ATP concentration. 1×MIC of COG1410 was added to the reaction tube, and samples were taken at 10 min, 30 min, and 60 min of reaction at 37 °C for ATP measurement. Ethambutol (EMB) was used as a control group in the experiment. It was repeated 3 times.

[0055] The extracellular ATP concentration was detected using an ATP detection kit (MAK190-1KT) from Merck, and the fluorescence generated was detected using an ELISA reader from BioTek at an excitation wavelength of 535 nm and an emission wavelength of 587 nm. For total ATP, a small amount of DMSO was added to the reaction system to disrupt the cell membrane, releasing all the intracellular ATP. The intracellular ATP concentration was obtained by subtracting the extracellular ATP from the total ATP, and the results are shown as Figure 4 described below.

[0056] As Figure 4 shown by the results, the amount of ATP leakage in the COG1410 treatment group increased with time.

[0057] Example 5

[0058] Frequency of induced resistance of COG1410

[0059] Synchronization was performed using the "synchronization" method for bacterial culture described in Example 3. The synchronized Mycobacterium abscessus strain standard strain ATCC19977 was inoculated into 2 mL of 7H9, and the OD was adjusted 600 to 0.05 as the starting point. 1 / 32 MIC of COG1410 was added to the 2 mL bacterial solution, and the mixture was cultured with shaking at 37 °C and 200 rpm for 24 h. No addition of antimicrobial peptide was used as a negative control, and addition of 0.5 μg / mL of rifampicin was used as a positive control. Subculture was performed every 24 h, and each time 20 μL of bacteria was transferred to 2 mL of fresh medium. After every 5 subcultures, the bacterial solution was collected and mixed with 20% sterile glycerol, and stored at -80 °C. Every 10 subcultures, the concentration of the antimicrobial substance in the medium was doubled. After 60 consecutive subcultures, the MICs of the bacterial solutions treated with COG1410 and rifampicin and the starting strain were measured. After measuring the MIC, the bacterial solution collected in the last generation was diluted and plated, and 10 single colonies were picked and cultured separately to measure the MIC. Then, the culture was expanded, the bacterial cells were collected, genomic DNA was extracted, and next-generation sequencing was performed. Compared with the genome of the starting strain, SNPs and indels of the mutant strains were detected to determine the possible resistance mechanisms of Mycobacterium abscessus to antimicrobial peptides.

[0060] As Figure 5 can be seen, Mycobacterium abscessus is not easily resistant to COG1410, but is resistant to rifampicin.

[0061] Example 6

[0062] Bactericidal activity test of COG1410 in 50% plasma

[0063] To determine the bactericidal activity of COG1410 in 50% plasma, after "synchronizing" the standard strain of Mycobacterium abscessus ATCC19977, it was cultured to the logarithmic growth phase (OD 600 = 0.5), and 1 mL of bacterial cells was collected, approximately 1×10 7 CFU. The bacterial cells were resuspended in PBS or normal human plasma (50% plasma) diluted 1:1 with PBS. In the resuspended solution, 2, 4, 8, 16, 32, 64, 128 μg / mL of COG1410 was added respectively, incubated at 37 °C for 2 h, and then the remaining CFU was counted by the dilution plating method. The concentration of the lowest COG1410 that killed 90% of the bacteria under the above conditions was defined as the LC 90 . The experiment was repeated 3 times.

[0064] From Figure 6 the results, it was shown that when the concentration of COG1410 was 128 μg / mL, the bactericidal rate reached 90%, so the LC 90 was 128 μg / mL.

[0065] Example 7

[0066] Interaction between COG1410 and antibiotics

[0067] To determine whether the antimicrobial peptide can be used in combination with other commonly used antibiotics, the standard strain ATCC19977 was selected for the experiment. The antibiotics selected were the first-line drugs for treating abscesses: rifampicin, kanamycin, ethambutol, amikacin, ciprofloxacin, clarithromycin, a total of 6 kinds. The effect of the combination of one antibiotic and COG1410 was tested each time.

[0068] 100 μL of drug A with a concentration of 16×MIC was added to the first column of a 96-well plate, 50 μL of 7H9 liquid medium containing 10% enrichment broth was added to the 2nd - 8th columns, 100 μL of medium was added to the 9th column, and 200 μL of medium was added to the 10th column; 50 μL of the liquid was aspirated from the first column to the second column, serially diluted to the 8th column in sequence, and then 50 μL of the liquid was aspirated and discarded from the 8th column; drug B was diluted in an Ep tube, and the concentration was diluted from 16×MIC to 1 / 4MIC and then added to rows A - G of the 96-well plate, and 50 μL of medium was added to row H; finally, 100 μL of a bacterial suspension with OD600 = 0.02 was added to A1 - H9 of the 96-well plate; the 96-well plate was placed in an incubator at 37 °C for 48 h, and then the absorbance at 600 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader and the antibacterial effect of the combination was calculated.

[0069] Result judgment: The fractional inhibitory concentration (FIC) index was used as the judgment criterion.

[0070] FICI index = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone.

[0071] In the above formula, drug A is COG1410 and drug B is the tested clinical antibiotic. Judgment criteria: FICI index < 0.5 indicates synergistic effect; 0.5 - 1 indicates additive effect; 1 - 2 indicates no relevant effect; > 2 indicates antagonistic effect.

[0072] Table 2 Results of the combination of COG1410 and antibiotics

[0073]

[0074] The results in Table 2 show that COG1410 can be combined with rifampicin, kanamycin, ethambutol, and amikacin respectively.

[0075] Example 8

[0076] Intracellular bactericidal experiment of COG1410 in a mouse macrophage phagocytosis model of Mycobacterium abscessus infection

[0077] Establish a mouse macrophage cell line RAW264.7 infection model. Seed mouse macrophages RAW264.7 in 24-well plates (2×10 5 cells / well), incubate overnight at 37°C and 5% CO2 for 20 h until the cells adhere and grow. Infect RAW264.7 with Mycobacterium abscessus ATCC19977 in the logarithmic growth phase at an MOI of 10:1. After 4 h of infection, add 1×MIC, i.e., 8 μg / mL of COG1410, and set up a control. The non-treatment group is the negative control. Samples are taken and diluted after administration and then spread on 7H10 agar plates, and cultured in an incubator at 37°C for 1 h, 6 h, 18 h, and 24 h, and the colony counts of different groups are calculated.

[0078] As Figure 7 can be seen, compared with the negative control group, COG1410 can effectively inhibit Mycobacterium abscessus.

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of Apo-E mimetic peptide COG1410 in the preparation of a drug for inhibiting mycobacteria, characterized in that, The mycobacteria mentioned above include one or more of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium abscessus; The Mycobacterium abscessus is Mycobacterium abscessus ATCC19977, the Mycobacterium tuberculosis is Mycobacterium tuberculosis H37Rv, and the Mycobacterium smegmatis is Mycobacterium smegmatis Mc 2 155.

2. The application according to claim 1, characterized in that The Apo-E mimetic peptide COG1410 disrupts the integrity of the mycobacterial cell membrane.

3. Use of the Apo-E mimetic peptide COG1410 in combination with an antibiotic for the preparation of a drug for inhibiting mycobacteria, characterized in that, The mycobacteria mentioned above include one or more of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium abscessus; The Mycobacterium abscessus is Mycobacterium abscessus ATCC19977, the Mycobacterium tuberculosis is Mycobacterium tuberculosis H37Rv, and the Mycobacterium smegmatis is Mycobacterium smegmatis Mc 2 155.

4. The application according to claim 3, wherein The antibiotics include one or more of rifampicin, kanamycin, ethambutol, amikacin, ciprofloxacin, and clarithromycin.

5. The application according to claim 3, characterized in that, The concentration of the Apo-E mimetic peptide COG1410 is 2 - 20 μg / mL, and the concentration of the antibiotic is 0.2 - 20 μg / mL.

6. The application according to any one of claims 1 and 3 to 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

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