Application of cyclic peptide compound BIM 23042 or preparation of cyclic peptide compound BIM 23042 in preparation of medicine for treating bacterial infectious diseases

Abstract

The invention discloses application of a cyclic peptide compound BIM 23042 or a preparation thereof in preparation of medicines for treating bacterial infectious diseases, and belongs to the technical field of medicines. The invention discloses a cyclic peptide compound BIM 23042 as an antibiotic synergist for the first time, and the cyclic peptide compound BIM 23042 is used for treating bacterial infectious diseases. According to the application disclosed by the invention, the cyclopeptide compound BIM 23042 and polymyxin are combined for use, so that the growth of gram-negative bacteria can be effectively and synergistically inhibited. The combined medication scheme shows a remarkable advantage in coping with acinetobacter baumannii. The cyclic peptide compound BIM 23042 disclosed by the invention has an anti-infection capability, and the anti-infection capability is specifically shown as reducing the inflammatory response of macrophage RAW 264.7 induced by LPS (Lipopolysaccharide) and also reducing the apoptosis of the macrophage RAW 264.7 caused by the LPS. The invention provides a new perspective for developing a novel drug-resistant gram-negative bacterium prevention and control strategy, and also provides a new treatment scheme for coping with bacterial infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a cyclic peptide compound BIM 23042 or a preparation thereof in preparation of a drug for a bacterial infectious disease. BACKGROUND

[0002] Acinetobacter baumannii is a gram-negative opportunistic pathogen that often causes severe infections in the respiratory tract, urinary tract, bloodstream, and wounds. Due to its strong environmental survival ability and natural multi-drug resistance, it has become one of the main pathogens of hospital-acquired infections. In particular, in patients in intensive care units, mechanical ventilation, and long-term antibiotic therapy, the incidence and mortality of Acinetobacter baumannii infection are significantly increased. In recent years, the rapid prevalence of multi-drug resistant (MDR) and pan-drug resistant (PDR) Acinetobacter baumannii has made the effective treatment drugs in the clinic increasingly scarce. Notably, Acinetobacter baumannii has shown increasing resistance to key drugs such as carbapenems and polymyxins, which seriously threatens global public health security. The World Health Organization (WHO) has listed Acinetobacter baumannii as one of the "highest priority" drug-resistant pathogens, highlighting the urgency and necessity of developing new anti-infective strategies.

[0003] Despite the continuous increase in global investment in the research and development of new antibacterial drugs, the number of completely new antibiotics approved in the past few decades remains limited, far from enough to cope with the increasingly severe drug resistance situation. Therefore, how to improve the efficacy of existing antibacterial drugs and extend their lifespan has become a research focus in the anti-infective field. In recent years, antibiotic combination or antibiotic potentiator therapy has been generally considered an effective way to break through the dilemma. This strategy not only restores the sensitivity of drug-resistant bacteria to antibiotics, but also enhances drug efficacy, reduces toxicity and side effects, shortens the course of treatment, and delays the emergence and spread of drug-resistant strains. Compared with the current situation of long development cycle, high investment, and high risk of new drug development, the combination of antibiotics and potentiators provides a more realistic and feasible solution to anti-infective therapy. Traditional antibiotics such as fosfomycin, tigecycline, and polymyxins have once again become the focus due to their synergistic potential in combination therapy and play an important role in dealing with multi-drug resistant bacterial infections.

[0004] The cyclic peptide compound BIM 23042, referred to as C4, has a molecular formula of C 65 H 77 N 11 O 11 S2, and a chemical structural formula as shown in formula 1:

[0005] Formula 1 The cyclic peptide compound BIM 23042 is a somatostatin (SS) octapeptide analogue, and is a selective neuropeptide neuromedin B receptor (NMB-R, BB1) antagonist.

[0006] There is no report on the application of the cyclic peptide compound BIM 23042 in the treatment of bacterial infectious diseases. SUMMARY

[0007] The technical problem solved by the present application is to provide the application of the cyclic peptide compound BIM 23042 or a preparation thereof in the preparation of a drug for bacterial infectious diseases.

[0008] The technical problem solved by the present application is to provide a pharmaceutical composition and the application thereof in the preparation of a drug or an antibiotic synergist for bacterial infectious diseases.

[0009] Technical solution: The present application provides the application of the cyclic peptide compound BIM 23042 or a preparation thereof in the preparation of a drug for bacterial infectious diseases.

[0010] The bacteria are gram-negative bacteria.

[0011] The gram-negative bacteria are Acinetobacter baumannii ATCC 19606.

[0012] The preparation further comprises an antibiotic.

[0013] The antibiotic is polymyxin.

[0014] The present application provides a pharmaceutical composition comprising the cyclic peptide compound BIM 23042 and polymyxin.

[0015] The concentration of the cyclic peptide compound BIM 23042 is 1-32 μg / mL.

[0016] The concentration of the polymyxin is 0.0039-0.125 μg / mL.

[0017] The mass ratio of the cyclic peptide compound BIM 23042 to the polymyxin is 256:1 ~ 8205:1.

[0018] The present application provides the application of the pharmaceutical composition in the preparation of a drug or an antibiotic synergist for bacterial infectious diseases.

[0019] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application first discloses the cyclic peptide compound BIM 23042 as an antibiotic synergist for treating bacterial infectious diseases. Specifically, the present application finds that the combination of the cyclic peptide compound BIM 23042 and polymyxin can effectively synergistically inhibit the growth of gram-negative bacteria. This combination scheme shows remarkable advantages in dealing with Acinetobacter baumannii. The cyclic peptide compound BIM 23042 of the present application has the ability to resist infection, specifically manifested as reducing the inflammatory response of macrophage RAW 264.7 induced by LPS, and also reducing the apoptosis of macrophage RAW 264.7 caused by LPS. The present application provides a new perspective for developing a new drug-resistant gram-negative bacteria prevention and control strategy, and also provides a new treatment scheme for dealing with bacterial infections. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Evaluation of C4 for enhancing the in vitro effectiveness of polymyxin against Acinetobacter baumannii ATCC19606, wherein CON means control. (A) Assessment of the synergistic inhibition of bacterial growth by C4 and polymyxin by checkerboard assay and fractional inhibitory concentration index (FICI), with a FICI value of 0.25, proving the synergistic effect of this combination on the growth inhibition of Acinetobacter baumannii ATCC19606. (B) Time-kill curve of C4-polymyxin combination against Acinetobacter baumannii ATCC 19606.

[0021] Figure 2 Evaluation of C4 for reducing LPS-induced inflammatory response of macrophage RAW 264.7, wherein CON means control. (A, B) Levels of pro-inflammatory cytokines (including IL-6, IL-22, TNF-a, IL-1b and IFN-g) and anti-inflammatory cytokines (including IL-4, IL-10) measured by ELISA (A) and RT-qPCR (B).

[0022] Figure 3 Evaluation of the in vivo safety of C4. (A) Experimental protocol. BALB / c mice received intraperitoneal injection of vehicle or C4 (30 mg / kg) for 14 days (n = 8 per group). (B) Hemolytic activity of C4 on red blood cells. (C) Survival rate of mice during treatment. (D, E) Body weight and serum biochemistry (ALT, AST, CREA, urea, Na+, K+, Cl-) at day 2 and day 14. (F) H&E staining of liver and kidney of mice.

[0023] Figure 4Figure legends for pneumonia and thigh infection mouse models treated with C4 and polymyxin. CON means PBS negative control. (A) Experimental design of neutropenic mouse models of lung and thigh infection (n = 8 per group). (B, C) Cytokine analysis in lung and thigh tissues of mice after combination therapy. (D, E) Bacterial load in lung and thigh muscle of mice treated with C4 plus polymyxin. (F, G) H&E staining shows reduced tissue damage, improved alveolar structure, and reduced inflammation in the combination group. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be further described below in conjunction with the drawings. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, a number of variations and improvements can also be made, which should be considered to belong to the protection scope of the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials used are commercially available from conventional biochemical reagent stores unless otherwise specified. In the following examples, the quantitative experiments were set up with three repeated experiments, and the results were averaged.

[0025] The LB broth medium used in the examples is an aqueous solution containing 10 g / L of tryptone, 5 g / L of yeast extract powder, and 10 g / L of NaCl; the LB solid medium is based on the LB broth medium with the addition of 15 g / L Agra; the MHB broth medium is an aqueous solution of 2 g / L of beef extract, 1.5 g / L of soluble starch, and 17.5 g / L of acid hydrolyzed casein.

[0026] The cyclic peptide compound BIM 23042 (referred to as C4) and polymyxin used in the examples were purchased from Tao Technology Co., Ltd.

[0027] C57 female mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0028] The strain Acinetobacter baumannii (A. baumannii) used in the examples was Acinetobacter baumannii ATCC 19606 from the article Tsubouchi T, Suzuki M, Niki M,, et al . Complete Genome Sequence of Acinetobacter baumannii ATCC 19606 T , a Model Strain of Pathogenic Bacteria Causing Nosocomial Infection. Microbiol Resour Announc . 2020 May 14;9(20):e00289-20.

[0029] Example 1 C4 enhances the in vitro effectiveness of polymyxin against Acinetobacter baumannii ATCC 19606 1. C4 and polymyxin were tested for synergistic activity against Acinetobacter baumannii ATCC 19606 using checkerboard method. The checkerboard method with two-fold serial dilutions of drugs (8 x 8) was used to assess synergy between C4 and polymyxin. After 18 hours of incubation with bacterial suspension (1.5 x 10 6 CFU / mL), the absorbance at 600 nm was measured using a microplate reader. Fractional inhibitory concentration index (FICI) was calculated from two biological replicates for each combination using the formula: FIC index = MIC (A combination) / MIC (A alone) + MIC (B combination) / MIC (B alone). FICI ≤ 0.5 indicates synergy, 0.5 < FICI ≤ 4 indicates no interaction, and FICI > 4 indicates antagonism. The experiment was repeated independently 3 times. Results are shown in Figure Figure 1 A, synergistic inhibition of bacterial growth by C4 and polymyxin was assessed by checkerboard assay and fractional inhibitory concentration index (FICI). The fractional inhibitory concentration index (FICI) value was 0.25. This combination demonstrated synergistic inhibition of growth of Acinetobacter baumannii ATCC 19606.

[0030] 2. C4 and polymyxin were tested for killing effect on Acinetobacter baumannii ATCC 19606 using plate count method. Overnight culture of Acinetobacter baumannii ATCC 19606 was diluted 1 : 1000 into fresh MHB to give an initial OD 600 of 0.05 and incubated at 37 °C with continuous shaking (200 rpm) for 4 hours. The culture was then treated with C4 (final concentration of 8 μg / mL), polymyxin (final concentration of 0.0312 μg / mL), or C4 (final concentration of 8 μg / mL) + polymyxin (final concentration of 0.0312 μg / mL) combination for 8 hours, respectively. Plate colony count method was used to determine bacterial counts at 0, 2, 4, and 8 hours. MHB with PBS was used as negative control. Each experiment was repeated 3 times. Results are shown in Figure Figure 1 B, C4 or polymyxin alone failed to kill Acinetobacter baumannii ATCC 19606, while the combination of the two was effective in killing Acinetobacter baumannii ATCC 19606.

[0031] The results indicate that neither C4 nor polymyxin alone could kill Acinetobacter baumannii ATCC 19606, but the combined use of both was effective in killing Acinetobacter baumannii ATCC 19606. The synergistic effect was best when the C4 concentration was 8 μg / mL and the polymyxin concentration was 0.0312 μg / mL, as evidenced by the OD values ​​in the checkerboard assay and growth curve assay. 600 The values ​​were the lowest, the number of CFUs counted on LB plates was significantly reduced, the amount of green fluorescent protein (live bacteria) in flow cytometry and co-microscopy results was reduced, and the bacteria ruptured and died in scanning and transmission electron microscopy.

[0032] Example 2: C4 alleviates LPS-induced inflammatory response in RAW 264.7 macrophages.

[0033] 1. The levels of cytokines (IL-4 (Cat. No. YJ064310), IL-6 (Cat. No. YJ063159), IL-10 (Cat. No. YJ037873), IL-22 (Cat. No. YT063138), TNF-a (Cat. No. YJ002095), IL-1 β (Cat. No. YJ106733), INF-γ (Cat. No. YJ002277)) were detected using ELISA kits (enzyme-linked, Shanghai, China). According to the standard in the instruction manual of each different cytokine detection kit, 50 μL of different concentrations of standard reagents were added to the standard wells, and 40 μL of sample dilution reagents and 10 μL of cell culture supernatant were added to the enzyme-coated plate. Horseradish peroxidase (HRP) conjugated reagent (100 μL) was added to the standard wells and sample wells. The reaction wells were sealed with a plate sealing film and incubated at 37°C for 60 min. The sealing film was removed, the liquid was discarded, and the plate was spun dry. Each well was filled with washing solution, left for 30 seconds, and then discarded, and this process was repeated five times. Color developing agent A (50 μL) and color developing agent B (50 μL) were added to each well. After incubation at 37°C for 10 min, 50 μL of stop solution was added to each well to terminate the reaction. The absorbance of each well was measured using an enzyme label instrument at a wavelength of 450 nm. The corresponding concentration was calculated from the standard curve according to the OD value of the sample well. Through calculation, the standard curve of IL-4 was X=(Y-0.1979) / 0.0967, the standard curve of IL-6 was X=(Y-0.2903) / 0.0534, the standard curve of IL-10 was X=(Y-0.3257) / 0.0574, the standard curve of IL-22 was X=(Y-0.2042) / 0.0678, the standard curve of TNF-a was X=(Y-0.2148) / 0.0496, the standard curve of IL-1 β was X=(Y-0.1905) / 0.0515, and the standard curve of INF-γ was X=(Y-0.0935) / 0.041. The results are shown in Table A, C4 dose-dependently alleviated LPS-induced inflammation in RAW 264.7 cells, as shown by the down-regulation of pro-inflammatory factors and the up-regulation of anti-inflammatory factors. Figure 2

[0034] ​2. Total RNA was extracted from RAW 264.7 cells using RNeasy Mini Kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. A total of 1000 ng of purified RNA was reverse-transcribed into cDNA using the RT Master Kit (Takara, Shiga, Japan). PCR reaction mixtures were prepared using TB Green Premix ExTaq™ (Vazyme) with the following system: Premix 10 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, cDNA 1 μL, ddH2O 8.2 μL. The amplification parameters were: initial denaturation at 95°C for 30 s, followed by 95°C denaturation for 5 s, 60°C denaturation for 30 s, 72°C for 45 s, for 40 cycles. The primer sequences are shown in Table 1. The 16S rRNA expression level was used as an internal reference, and the comparative CT method was used to analyze the changes in the expression of other genes. The results are shown in Figure 2 Fig. B, C4 dose-dependently attenuated LPS-induced inflammation in RAW 264.7 cells, as evidenced by down-regulation of pro-inflammatory factors and up-regulation of anti-inflammatory factors.

[0035] Table 1. RT-PCR primer sequences

[0036] Example 3 In vivo safety evaluation of C4 The hemolysis rate of C4 was examined using sterile sheep red blood cells. Briefly, 8% sheep red blood cells (Qingdao Haibo, Catalog No. 1001339) were prepared and incubated with serial concentrations of C4 (0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 μg / mL) at 37°C for 1 hour, with sterile PBS as a blank control and ddH2O as a positive control. After incubation, the absorbance of the supernatant at 576 nm was detected using an Infinite M200 enzyme marker (Tecan), and the hemolysis rate was calculated according to the controls. The results are shown in Figure 3 As shown in Fig. B, high concentrations of C4 (512 μg / mL) did not cause hemolysis of sheep red blood cells compared to the control group.

[0037] 32 seven-week-old BALB / c male mice (22-25 g) were used for the safety evaluation of C4. These mice were randomly divided into two groups, 8 mice in each group, C4 was 30 mg / kg, intraperitoneal injection of DMSO as negative control group. Body weight was recorded during normal feeding for 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, blood (1 mL) was collected from the mice by enucleation. Serum biochemical indicators include alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA) and serum creatinine (CREA). After 14 days of treatment, the levels of sodium (Na + ), potassium (K + ) and chloride (Cl − ) in the blood were analyzed. 14 days after inoculation, the mice were euthanized by cervical dislocation, and the liver and kidney were collected for histopathological slides according to the standard staining procedure. The results are shown in Figure 3 C-F, there was no significant difference in body weight in the C4 treatment group, the survival rate was 100%, there was no significant difference in ALT, AST, UREA, CREA detection, there was no difference in Na + , K + , CL - content, and they were all within the normal range, and there was no significant difference in liver and kidney HE staining results, indicating that C4 showed good in vivo safety in mice.

[0038] Example 4 C4 and polymyxin combination therapy of mouse pneumonia and mouse thigh infection Female ICR six-week-old mice (n = 8 per group, body weight 20-22 g) purchased from the Comparative Medicine Center of Yangzhou University were first neutrophil-reduced by cyclophosphamide (Macklin Company) (150 mg / kg and 100 mg / kg for two consecutive days 4 days and 1 day before infection). Using a P50~P200 pipette (Thermo Fisher), 50 μL of P. aeruginosa ATCC 19606 (1×10 8 CFU per mouse) suspension was applied to the nostrils when the animals inhaled the bacterial suspension. Two hours after infection, C4 (5 mg / kg) or polymyxin (2.5 mg / kg) was given alone or in combination (C4 5 mg / kg + polymyxin 2.5 mg / kg) by intraperitoneal injection. Four hours after infection, the mice were euthanized by cervical dislocation. The lungs were aseptically removed, homogenized, serially diluted, and inoculated on LBA plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar) and incubated at 37 °C for 24 h before counting the number of bacteria. At the same time, the levels of pro-inflammatory and anti-inflammatory factors in the lung tissue were detected by the method in Example 2 above, and the results are shown in Figure 4As shown in B and D, compared with the control group and the group using C4 or polymyxin alone, the levels of pro-inflammatory cytokines (including IL-6, IL-22, TNF-a, IL-1b and IFN-g) in the lung tissues of mice were significantly reduced and the levels of anti-inflammatory cytokines (including IL-4, IL-10) were significantly increased, while the bacterial load in the lungs of mice was reduced.

[0039] Female ICR mice (n = 8 per group) were first neutropenic by cyclophosphamide (150 and 100 mg / kg administered twice in a row 4 days and 1 day before infection). Then, 100 μL of Acinetobacter baumannii ATCC 19606 bacterial suspension (1.0 x 10 7 CFU) was injected into the right thigh of each mouse. 2 hours after infection, C4 (5 mg / kg) or polymyxin (2.5 mg / kg) was administered alone or in combination (C4 5 mg / kg + polymyxin 2.5 mg / kg) by intraperitoneal injection. 24 hours after infection, the mice were euthanized by cervical dislocation. The right thigh muscle was aseptically removed, homogenized, serially diluted, inoculated on LBA plates, and after incubation at 37°C, the number of bacteria was counted. At the same time, the levels of pro-inflammatory and anti-inflammatory factors in the thigh muscle tissues were detected by the method in Example 2 above, and the results are shown in Figure 4 As shown in C and E, compared with the control group and the group using C4 or polymyxin alone, the levels of pro-inflammatory cytokines (including IL-6, IL-22, TNF-a, IL-1b and IFN-g) in the thigh muscle tissues of mice were significantly reduced and the levels of anti-inflammatory cytokines (including IL-4, IL-10) were significantly increased, while the bacterial load in the thigh muscle of mice was reduced.

[0040] As shown in F and G in Figure 4 , H&E staining showed that the lung and thigh muscle tissue damage and inflammation of the combination group were reduced.

[0041] In summary, C4 is a potential polymyxin potentiator that can effectively enhance the in vitro and in vivo antibacterial effect of polymyxin on Acinetobacter baumannii ATCC 19606.

Claims

1. Use of the cyclic peptide BIM 23042 or a formulation thereof for the preparation of a medicament for the treatment of a bacterial infectious disease.

2. Use according to claim 1, characterized in that, The bacteria are Gram-negative bacteria.

3. Use according to claim 2, characterized in that, The Gram-negative bacteria are Acinetobacter baumannii ATCC 19606.

4. Use according to claim 1, characterized in that, The formulation also comprises an antibiotic.

5. Use according to claim 4, characterized in that, The antibiotic is a polymyxin.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the cyclic peptide BIM 23042 and a polymyxin.

7. The pharmaceutical composition of claim 5, wherein, The concentration of the cyclic peptide BIM 23042 is between 1 and 32 μg / mL.

8. The pharmaceutical composition of claim 5, wherein, The concentration of the polymyxin is between 0.0039 and 0.125 μg / mL.

9. The pharmaceutical composition of claim 5, wherein, The mass ratio between the cyclic peptide BIM 23042 and the polymyxin is between 256:1 and 8205:

1.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 for the preparation of a medicament for the treatment of a bacterial infectious disease or an antibiotic potentiator.

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