CN107236022A - Lipophilic compound conjugates of cell-penetrating peptides and their application in antibacterial - Google Patents

Abstract

The invention discloses a lipophilic compound conjugate of a cell penetrating peptide and application thereof in antibiosis. The lipophilic compound conjugate of the cell penetrating peptide is formed by connecting the cell penetrating peptide and a lipophilic compound connected with the amino terminal or the carboxyl terminal of the cell penetrating peptide. The lipophilic compound conjugate of the antibacterial peptide has good inhibiting and killing effects on gram-positive bacteria, gram-negative bacteria and fungi, and has broad-spectrum antibacterial effect. Wherein, in the aspect of inhibiting methicillin-resistant staphylococcus aureus, the C12-TAT and clarithromycin have additive effect; the C12-TAT has obvious synergistic effect in combination with imipenem. In the aspect of inhibiting pseudomonas aeruginosa, C16-TAT has an additive effect in combination with clarithromycin; the combination of C16-TAT and imipenem has obvious synergistic effect.

Description

Lipophilic compound conjugates of cell-penetrating peptides and their application in antibacterial

technical field

The invention relates to a lipophilic compound conjugate of a cell penetrating peptide in the field of antibacterial and its application in antibacterial.

Background technique

Antibiotics have always been the main drug for clinical anti-infection treatment. The great success of antibiotics from the 1940s to the 1970s has reduced the attention and investment in the research and development of antibacterial drugs. In the past 40 years, the research and development of anti-drug-resistant bacteria in the world has been slow. Mechanisms and drugs with new targets are rarely launched, and known effective drugs (or their derivatives) have been repeatedly used or even abused for many years, resulting in a decline in drug efficacy and drug resistance.

The growing problem of bacterial resistance to antibiotics has received widespread attention worldwide. Due to the continuous emergence of resistant strains of various antibacterial drugs, the trend of multi-drug resistance has gradually expanded, which has had a profound impact on clinical medication, making drug resistance a new problem in the field of health. In the 1960s, the number of people who died of infectious diseases in the world was about 7 million per year, and it rose to 20 million at the beginning of this century; the number of people who died of sepsis rose by 89%, most of them died of super drug-resistant "super bacteria". "Bringing medication difficulties. WHO established the theme of World Day 2011 as "Controlling Antimicrobial Resistance: No Action Today, No Drugs Available Tomorrow", which once again proved the seriousness of bacterial resistance.

In particular, multidrug-resistant and pan-drug-resistant Gram-negative bacteria are increasing year by year, which has become a difficulty in anti-infection treatment. Compared with Gram-positive bacteria, Gram-negative bacilli (GNB) have attracted special attention because they are prone to acquire or up-regulate resistance-related genes under the selective pressure of antibiotics. According to the US National Medical Safety Network data, GNB accounts for more than 30% of hospital-acquired infection pathogens and is the most common pathogen in respiratory-associated pneumonia and urinary tract infection. In 2010, the New Delhi metallo-beta-lactamase type I (NDM-1) Gram-negative bacteria that caused widespread drug resistance appeared simultaneously in many regions of the world, which once again sounded the alarm. These increasing bacterial drug resistance problems and increasingly complex drug resistance mechanisms have brought great difficulties to the treatment of related diseases, and have even seriously endangered the health and lives of patients.

At the same time, the resistance of bacteria existing in the form of biofilm to antibacterial drugs, external environmental pressure and host immune system is significantly enhanced, which is also one of the important reasons leading to bacterial drug resistance and difficult treatment of infectious diseases. Bacterial biofilm is a highly organized and systematic bacterial community that is wrapped in extracellular matrix composed of extracellular polysaccharides, proteins, nucleic acids, etc. secreted by bacteria, adheres to each other, and grows on a certain carrier surface. Biofilms can be formed by one or a mixture of several bacteria. Biofilm is a growth mode corresponding to planktonic bacteria formed during the growth process of bacteria to adapt to the living environment. It shows a series of new biological characteristics different from planktonic bacteria on the whole, and has stronger adaptability. external environment capabilities. The resistance of biofilm bacteria to antibiotics is more than 1000 times that of planktonic bacteria, and its drug resistance is mostly due to the multicellular structure of biofilm. According to preliminary statistics from the National Institutes of Health (NIH), 80% of bacterial diseases are related to bacterial biofilms. Bacterial biofilms are widely present on the Therefore, finding anti-drug resistance strategies that can simultaneously target drug-resistant bacteria and drug-resistant bacterial biofilms has become a major breakthrough in combating bacterial resistance, especially Gram-negative bacteria.

The use of antibiotics and antibacterial drugs is the most basic and most commonly used method to control bacterial biofilm infection. However, with the increase of bacterial drug resistance, while developing effective new antibacterial drugs, some new active substances different from traditional antibacterial drugs and New treatments are also beginning to be used in the field of antibacterial biofilm research.

Cell penetrating peptides (CPPs) are a class of short peptides (generally less than 35 amino acid residues) that can enter cells through various cell membranes. Its discovery originated in 1988, when some scholars discovered that HIV-1's transactivator protein Tat can be transduced into cells across the membrane, and then someone discovered that the Drosophila transcriptional protein also has similar characteristics. Scholars have obtained the shortest peptide sequences required for transduction from these transcription factors and found that they can introduce other macromolecules (such as proteins, nucleic acids, etc.) into cells. Since then, many other CPPs have been discovered one after another. Table 1 lists some representative CPPs.

Table 1 Its representative CPPs

Common clinical Gram-positive pathogenic bacteria include: Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis), Streptococcus pyogenes, Streptococcus pneumoniae , Enterococcus faecalis, Enterococcus faecium, Mycobacterium tuberculosis (MTB), Bacillus subtilis, Bifidobacterium breve and Bifidobacterium longum ( Bifidobacterium longum). Among them, Staphylococcus aureus includes Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus pneumoniae includes penicillin-resistant Streptococcus pneumoniae Penicillin-resistant streptococcus pneumoniae (PRSP) and penicillin-sensitive streptococcus pneumoniae (PSSP), Enterococcus faecalis including vancomycin-resistant enterococcus faecalis (VRE) and vancomycin-sensitive Enterococcus (Vancomycin-sensitive enterococcus faecium, VSE), Enterococcus faecium includes vancomycin-resistant enterococcus faecium (VRE) and vancomycin-sensitive enterococcus faecium (VSE). Staphylococcus aureus and Staphylococcus epidermidis belong to the bacterial kingdom Firmicutes (Firmicutes), Bacilli (Bacilli), Bacillales (Bacillales), Staphylococcus (Staphylococcus). Enterococcus faecalis and Enterococcus faecium belong to the bacterial kingdom Firmicutes phylum Bacillus class Lactobacillales (Lactobacillales) Enterococcus family (Enterococcaceae) Enterococcus (Enterococcus). Streptococcus pyogenes and Streptococcus pneumoniae belong to the bacterial kingdom Firmicutes phylum Bacillus class Lactobacillus order Streptococcus (Streptococcaceae) Streptococcus (Streptococcus). Mycobacterium tuberculosis belongs to the bacterial kingdom Firmicutes, Schizomycetes, Actinomycetales, Mycobacteriaceae, Mycobacterium. Bacillus subtilis belongs to the bacterial kingdom Firmicutes Bacillus class Bacillus order Bacillus family (Bacillaceae) Bacillus (Bacillus). Bifidobacterium breve and Bifidobacterium longum belong to the bacterial kingdom Actinobacteria (Actinobacteria) Actinobacteria (Actinobacteria) (Actinobacteridae) Bifidobacteriales (Bifidobacteriales) Bifidobacteriaceae (Bifidobacteriaceae) Bifidobacteria Bacillus (Bifidobacterium).

Common clinical Gram-negative pathogenic bacteria include: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacter cloacae, Citrobacter diversus, Morganella morganii, Haemophilus influenzae, Parainfluenzae Haemophilus parainfluenzae and Legionella pneumophila. Among them, Escherichia coli belongs to the phylum Proteobacteria, the class Gammaproteobacteria, the order Enterobacteriales, the family Enterobacteriaceae, and the genus Escherichia. Klebsiella pneumoniae belongs to the bacterial kingdom Proteobacteria γ-Proteobacteria Enterobacteriaceae Enterobacteriaceae Klebsiella (Klebsiella). Acinetobacter baumannii belongs to the bacterial kingdom Proteobacteria γ-Proteobacteria Pseudomonales (Pseudomonadales) Moraxellaceae (Moraxellaceae) Acinetobacter (Acinetobacter). Pseudomonas aeruginosa belongs to the bacterial kingdom Proteobacteria γ-proteobacteria Pseudomonas order Pseudomonas (Pseudomonadaceae) Pseudomonas (Pseudomonas). Stenotrophomonas maltophilia belongs to the bacterial kingdom Phylum BX11. Proteobacteria Gamma Proteobacteria Gammaproteobacteria Xanthomonadales Genus (Stenotrophomonas). Enterobacter cloacae belongs to the bacterial kingdom Proteobacteria γ-proteobacteria Enterobacteriaceae Enterobacteriaceae (Enterobacter). Citrobacter heterotype belongs to the bacterial kingdom Proteobacteria γ-Proteobacteria Enterobacteriaceae Enterobacteriaceae Citrobacter (Citrobacter). Morganella morganii belongs to the bacterial kingdom Proteobacteria γ-Proteobacteria Enterobacteriaceae Enterobacteriaceae Morganella (Morganella). Haemophilus influenzae belongs to the bacterial kingdom Proteobacteria γ-Proteobacteria Pasteurellales (Pasteurellales) Pasteurellaceae (Pasteurellaceae) Haemophilus (Haemophilus). Haemophilus parainfluenzae belongs to the bacterial kingdom Proteobacteria γ-proteobacteria Pasteurella order Pasteurella family Haemophilus genus. Legionella pneumophila belongs to the bacterial kingdom Proteobacteria phylum γ-proteobacteria Legionella order Legionella family Legionella (legionella).

Common clinical pathogenic fungi include: Candida albicans, Candida tropicalis, Candida parapsilosis, Aspergillus flavus and Aspergillus fumigatus. Among them, Candida albicans, Candida tropicalis and Candida parapsilosis belong to the fungal kingdom Fungal phylum Deuteromycotina subphylum Bacillus class Cryptococcus yeast order Cryptococcus family Candida genus. Aspergillus flavus and Aspergillus fumigatus belong to the fungal kingdom Fungi phylum Deuteromycetes subphylum Deuteromycetes class Shell mold order Calicinomyceae Aspergillus genus.

Contents of the invention

The technical problem to be solved by the invention is how to enhance the antibacterial activity of antibacterial drugs.

In order to solve the above technical problems, the present invention provides lipophilic compound conjugates of cell penetrating peptides (ie, lipopeptides) or pharmaceutically acceptable salts thereof.

The lipopeptide provided by the present invention is formed by linking a cell penetrating peptide with a lipophilic compound connected to the amino terminus or carboxyl terminus of the cell penetrating peptide.

Among the above-mentioned lipopeptides, the cell-penetrating peptide is the polypeptide of a) or b):

a) a polypeptide whose amino acid sequence is SEQ ID No.1;

b) a derivative polypeptide obtained by subjecting the polypeptide of a) to substitution and/or deletion and/or addition of 1 to 5 amino acid residues, and the derivative polypeptide has bactericidal activity and/or bacteriostatic activity.

Among the above lipopeptides, the lipophilic compound may be fatty acid or cholesterol, sphinganine or vitamin E, etc.

In the above-mentioned lipopeptides, the fatty acid can be F1, F2 or F3:

F1, the fatty acid is a saturated fatty acid or an unsaturated fatty acid;

F2, the fatty acid is a saturated fatty acid containing 8 to 20 carbon atoms;

F3, the fatty acid is caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18) or arachidic acid (C20).

Wherein, the polypeptide whose amino acid sequence is SEQ ID No.1 is a cell-penetrating peptide named TAT.

The lipopeptides can specifically be the five lipopeptides named C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, and C8-TAT is formed by linking TAT to C8 at the amino terminal of TAT. C12-TAT is formed by connecting TAT and C12 connected to the amino terminal of TAT, C14-TAT is formed by connecting TAT and C14 connected to the amino terminal of TAT, and C16-TAT is formed by connecting TAT and C16 connected to the amino terminal of TAT C20-TAT is formed by linking TAT and C20 connected to the amino terminus of TAT.

In the lipopeptide above, all amino acids in the cell penetrating peptide are L-type amino acids.

The lipopeptide pharmaceutical salt of the present invention can be formed by cations such as sodium, potassium, aluminum, calcium, lithium, manganese and zinc, etc., and can also be formed by bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, etc. acid, arginine, ornithine, choline, etc.

Derivatives of the above-mentioned lipopeptides also belong to the protection scope of the present invention.

The lipopeptide derivatives provided by the present invention can be the following 1), 2) or 3):

1) Using one or more amino acid residues in the cell penetrating peptide of the lipopeptide with a conformation of D-type amino acid residues, artificially modified amino acid residues and/or rare amino acid residues existing in nature Substitution is performed to obtain a derivative polypeptide;

2) a linker obtained by connecting an amino-terminal protecting group to the amino-terminus of the cell-penetrating peptide of the lipopeptide and/or connecting a carboxy-terminal protecting group to the carboxyl-terminus of the cell-penetrating peptide;

3) A linker in which an amino-terminal protecting group is connected to the amino-terminal of the lipopeptide derivative described in 1) and/or a carboxyl-terminal protecting group is connected to the carboxyl-terminal of the lipopeptide derivative described in 1).

Among the above-mentioned lipopeptide derivatives, D-type amino acids refer to amino acids corresponding to L-type amino acids that make up proteins; artificially modified amino acids refer to common L-type amino acids that make up proteins that have been modified by methylation, phosphorylation, etc. ; Rare amino acids that exist in nature include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, homoserine, etc.

The lipopeptide multimer of the following PM1 or PM2 also belongs to the protection scope of the present invention:

PM1, a multimer formed by said lipopeptide or a pharmaceutically acceptable salt of said lipopeptide;

PM2, a multimer formed by derivatives of said lipopeptide.

In order to solve the above technical problems, the present invention provides the lipopeptide or the pharmaceutically acceptable salt of the lipopeptide or the derivative of the lipopeptide or the multimer of the lipopeptide in the preparation of enhanced antimicrobial bactericidal activity and/or or antibacterial active products (drugs, antibacterial materials or micellar nanoparticles).

In the above application, the antibacterial drug is a drug with bactericidal activity and/or antibacterial activity, including various antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones and other chemically synthesized drugs, and also includes bacteria, radioactive Some products obtained by culturing microorganisms such as nematodes and fungi, or the same or similar substances produced by chemical semi-synthesis. The antibacterial drug can specifically be a drug containing imipenem or/and clarithromycin.

In the above application, the active ingredient of the antibacterial drug can be imipenem or/and clarithromycin, and the active ingredient of the antibacterial drug can also contain the lipopeptide or the pharmaceutically acceptable salt of the lipopeptide or the Derivatives of the lipopeptide or other components other than the multimer of the lipopeptide, other active ingredients of the antibacterial drug can be determined by those skilled in the art according to the antibacterial effect.

Following P1 or P2 also belong to protection scope of the present invention:

P1, an antibacterial product comprising M1) and M2); said M1) is M11) or/and M12) or/and M13); said M11) is said lipopeptide or a pharmaceutically acceptable salt of said lipopeptide; The M12) is a derivative of the lipopeptide; the M13) is a multimer of the lipopeptide;

The M2) is the antibacterial drug;

P2, antibacterial product, it comprises M1) described in P1.

Wherein, the antibacterial product described in P1 and the antibacterial product described in P2 can be a drug with bactericidal activity and/or bacteriostatic activity, a coating with bactericidal activity and/or bacteriostatic activity, or a coating with bactericidal activity and/or bacteriostatic activity. Material. Wherein, the coating having bactericidal activity and/or bacteriostatic activity may be an antibacterial coating used on the surface of an implant implanted into an animal body.

In the antibacterial product described in P1, in the drug with bactericidal activity and/or bacteriostatic activity, the M1) and the M2) can be mixed together, and the M1) and the M2) can also be packaged independently To form a set of drugs, the M2) can also be entrapped in the M1) to form micellar nanoparticles. In the medicament with bactericidal activity and/or bacteriostatic activity, the mass ratio of the M1) to the M2) may be 1:1-10:1 (such as 1:1).

In the antibacterial product described in P1, the active ingredient of the antibacterial product described in P1 can be the M1) and the M2), the active ingredient of the antibacterial product described in P1 can also contain other ingredients, and the other active ingredients of the antibacterial product described in P1 Those skilled in the art can determine according to the antibacterial effect.

In the antibacterial product described in P2, the active ingredient of the antibacterial product described in P2 can be said M1), the active ingredient of the antibacterial product described in P2 can also contain other ingredients, and other active ingredients of the antibacterial product described in P2 can be obtained by those skilled in the art According to the antibacterial effect to determine.

The antibacterial product described in P1 and the antibacterial product described in P2 may also contain pharmaceutically acceptable carriers or excipients. The carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials Materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.).

The application of the M1) or the M1) and the M2) in the preparation of the P1 or P2 also belongs to the protection scope of the present invention.

Herein, the bactericidal activity and/or bacteriostatic activity have killing and/or inhibitory effect on the following pathogenic bacteria described in B1 to B23:

B1, Gram-positive bacteria;

B2, Firmicutes bacteria or Actinomycetes bacteria;

B3, bacteria of the class Bacillus, bacteria of the class Schizobacter or bacteria of the class Actinomycetes;

B4. Bacillus, Lactobacillus, Actinomycetes or Bifidobacteria;

B5. Staphylococcus, Enterococcus, Streptococcus, Mycobacteria, Bacillus or Bifidobacteria;

B6. Staphylococcus bacteria, Enterococcus bacteria, Streptococcus bacteria, Mycobacterium bacteria, Bacillus bacteria or Bifidobacterium bacteria;

B7, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Mycobacterium tuberculosis, Bacillus subtilis, Bifidobacterium breve or Bifidobacterium longum;

B8, Methicillin-resistant Staphylococcus aureus, Methicillin-sensitive Staphylococcus aureus, Vancomycin-resistant Enterococcus faecalis, Vancomycin-sensitive Enterococcus faecalis, Vancomycin-resistant Enterococcus faecium, Vancomycin-sensitive Enterococcus faecium cocci, penicillin-resistant Streptococcus pneumoniae, or penicillin-sensitive Streptococcus pneumoniae;

B9. Enterococcus faecalis resistant to vancomycin, gentamicin and streptomycin, Enterococcus faecium resistant to vancomycin and teicoplanin, Streptococcus pneumoniae resistant to cephalosporin and penicillin;

B10, Gram-negative bacteria;

B11, Proteobacteria or Proteus bacteria;

B12, γ-Proteobacteria, Proteus bacteria;

B13, bacteria of the order Enterobacteriaceae, bacteria of the order Pseudomonas, bacteria of the order Xanthomonas, bacteria of the order Pasteurella or bacteria of the order Legionella;

B14, Enterobacteriaceae, Moraxellaceae, Pseudomonases, Xanthomonasceae, Pasteurellaceae or Legionellaceae;

B15, Escherichia bacteria, Klebsiella bacteria, Acinetobacter bacteria, Pseudomonas bacteria, Stenotrophomonas bacteria, Enterobacter bacteria, Citrobacter bacteria, Bacteria of the genus Morganella, Haemophilus or Legionella;

B16, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacter cloacae, Citrobacter heterotype, Morganella morganii, Influenzaphila Haemophilus, Haemophilus parainfluenzae, or Legionella pneumophila;

B17. Fungi;

B18, Deuteromycotina fungi;

B19, Bacillus fungi or Deuteromycetes fungi;

B20. Fungi of the order Cryptococcus or fungi of the order Shellomyces;

B21, Cryptococcomycetes fungi or Calicinomycetes fungi;

B22, Candida fungi or Aspergillus fungi;

B23, Candida albicans, Candida tropicalis, Candida parapsilosis, Aspergillus flavus or Aspergillus fumigatus.

The present invention also provides pharmaceutical compounds.

The pharmaceutical compound provided by the present invention is the lipopeptide or the pharmaceutically acceptable salt of the lipopeptide, or the derivative of the lipopeptide; or the multimer of the lipopeptide.

The pharmaceutical compound can be the above-mentioned drugs with bactericidal activity and/or bacteriostatic activity.

The present invention also provides a method for treating or/and preventing pathogenic bacteria from infecting animals, comprising administering said M1) or said P1 or said P2 to recipient animals to inhibit pathogenic bacteria from infecting animals.

The pathogenic bacteria are bacteria and/or fungi, such as any one of the B1 to B23.

In practical application, the lipopeptide or its pharmaceutically acceptable salt, the derivative, the multimer, and the pharmaceutical compound of the present invention can be directly administered to the patient as a drug, or combined with a suitable carrier or excipient After being mixed, antibacterial drugs are prepared and given to patients to achieve the purpose of treating and/or preventing pathogenic bacteria infections.

Experiments have shown that C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, especially C12-TAT, C14-TAT, C16-TAT and C20-TAT are effective against Gram-positive bacteria, Gram Both negative bacteria and fungi have good inhibitory and killing effects, and have broad-spectrum antibacterial effects. Among them, C12-TAT has good dispersion effect on methicillin-resistant Staphylococcus aureus (MRSA) biofilm, and C16-TAT has good dispersion effect on Pseudomonas aeruginosa (P.aeruginosa) biofilm, and has antibacterial biofilm effect. In inhibiting methicillin-resistant Staphylococcus aureus, C12-TAT combined with clarithromycin has an additive effect; C12-TAT combined with imipenem has an obvious synergistic effect. In inhibiting Pseudomonas aeruginosa, C16-TAT combined with clarithromycin has an additive effect; C16-TAT combined with imipenem has an obvious synergistic effect. C12-TAT and C16-TAT have good antibacterial activity in vivo, which is equivalent to or slightly better than that of antibacterial drugs imipenem and clarithromycin at the same dose. Efficacy of C12-TAT combined with imipenem on lung infection and skin infection caused by methicillin-resistant Staphylococcus aureus, C16-TAT combined with imipenem on lung infection and skin infection caused by Pseudomonas aeruginosa The curative effect is significantly better than the curative effect of the two alone. It shows that C12-TAT combined with imipenem and C16-TAT combined with imipenem not only have a synergistic effect on drug-resistant bacteria in vitro, but also have better efficacy than single administration in terms of anti-drug-resistant bacterial infection in animals.

Description of drawings

Figure 1 is the reaction formula for preparing lipopeptides.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation of lipopeptide

In this example, six lipopeptides named C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT were prepared, all of which were penetrated by cells named TAT Peptide (amino acid sequence is YGRKKRRQRRR ie SEQ ID No. 1), which is formed by linking lipophilic compound connected to the amino terminus of the cell penetrating peptide.

The structural formula of C4-TAT is: C4-YGRKKRRQRRR, wherein C4 is butyric acid;

The structural formula of C8-TAT is: C8-YGRKKRRQRRR, wherein C8 is octanoic acid;

The structural formula of C12-TAT is: C12-YGRKKRRQRRR, wherein C12 is lauric acid;

The structural formula of C14-TAT is: C14-YGRKKRRQRRR, wherein, C14 is myristic acid;

The structural formula of C16-TAT is: C16-YGRKKRRQRRR, wherein, C16 is palmitic acid;

The structural formula of C20-TAT is: C20-YGRKKRRQRRR, wherein C20 is arachidic acid;

YGRKKRRQRRR in the structural formulas of C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT are all L-type amino acids.

Its synthesis method is as follows:

Saturated fatty acids with different carbon chain lengths were dissolved in acetonitrile respectively, activated with EDC and NHS respectively, and then reacted with TAT (at a molar ratio of 1:1) at low temperature and dark conditions, the specific reaction formula is shown in Figure 1 . In this reaction formula, CH ₃ (CH ₂ ) _n COOH represents butyric acid, caprylic acid, lauric acid, myristic acid, palmitic acid or arachidic acid.

The solvent was removed by rotary evaporation of the product, unreacted raw materials and by-products were removed by column separation, the reaction conversion rate was determined by HPLC, and the structure of the product was confirmed by NMR, MS, and IR. The results showed that C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT with the above structural formulas were obtained with a purity greater than 95%.

Embodiment 2, the antibacterial action of lipopeptide

1. Determination of minimum inhibitory concentration (MIC)

Adopt plate double dilution method to carry out drug susceptibility test, measure the minimum inhibitory concentration (MIC) of C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT in embodiment 1, specifically The experimental method is as follows: the test bacteria (as shown in Table 2) were cultured with MH broth medium. Drugs (C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT in Example 1) are diluted into various required concentrations with MH broth medium twice, and an appropriate amount is added respectively Put the MH agar medium into the plate, after the MH agar medium is melted, pour it quantitatively into the plate containing the drug solution and mix it evenly, inoculate various microorganisms to be tested with a multi-point inoculator (the inoculation amount is 10 ⁴ CFU/point), and then place it in a 37°C incubator to keep the temperature Observe the results after culturing for 18 hours, the minimum concentration of the drug contained in the aseptically grown plate is the minimum inhibitory concentration (Minimal Inhibitory Concentration, MIC).

The result is as follows:

Table 2. MIC values of TAT and lipopeptides on each bacteria (μg/mL)

The results showed that C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, especially C12-TAT, C14-TAT, C16-TAT and C20-TAT were effective against Gram-positive bacteria, Gram Both negative bacteria and fungi have good inhibitory and killing effects, and have broad-spectrum antibacterial effects.

In Table 2, MRSA refers to methicillin-resistant Staphylococcus aureus, VRE refers to vancomycin-resistant Enterococcus, PSSP refers to penicillin-sensitive Streptococcus pneumoniae, and PRSP refers to penicillin-resistant Streptococcus pneumoniae; Enterococcus faecalis ATCC51575 is resistant to vancomycin, Gentamicin and streptomycin were resistant, Enterococcus faecium ATCC700221 was resistant to vancomycin and teicoplanin, and Streptococcus pneumoniae ATCC51915 was resistant to cephalosporin and penicillin.

2. In vitro anti-biofilm effect evaluation

2.1 Establishment of in vitro biofilm model

The colony of Staphylococcus aureus ATCC33591 (MRSA) or Pseudomonas aeruginosa ATCC 27853 was picked and placed in 5mL MH medium, and incubated at 37°C with constant temperature and shaking. After 8 hours, the concentration of the bacterial solution was adjusted to 1.5×10 ⁸ CFU/mL with a turbidimeter, and diluted 100 times. Aspirate 2mL of the above bacterial solution, add it to a 24-well plate with 8×8mm glass slides, and incubate at a constant temperature of 37°C to form a mature biofilm.

2.2 Quantitative detection of biofilm growth status by crystal violet staining

Add different concentrations of drug solutions (the drugs refer to TAT, C12-TAT and C16-TAT in Example 1 respectively, and the solvents are all PBS solutions with a pH of 7.2) in each well to add an equivalent amount of 7.2 PBS solution was used as a blank control, and incubated at a constant temperature of 37°C for 24 hours and 48 hours. After 24 hours and 48 hours, gently rinse the 24-well plate with PBS solution and let it dry. Add 1% crystal violet solution to the orifice plate, stain for 20 minutes, rinse the orifice plate with PBS solution 3 times, and let it stand to dry. Add 95% ethanol to the orifice plate, let it stand for 15 minutes, elute the crystal violet, measure the absorbance value at 570nm wavelength with a microplate reader (the lower the value, the better the biofilm resolution effect), and use 95% ethanol as a control. And statistically analyze the data. The results are shown in Table 3-6:

Table 3. The dispersion situation (OD570nm) of Staphylococcus aureus ATCC33591 (MRSA) biofilm after adding medicine 24h

Table 4. Dispersion (OD570nm) of Staphylococcus aureus ATCC33591 (MRSA) biofilm after dosing 48h

concentration TAT C12-TAT 4MIC 0.957±0.025 0.500±0.010 2MIC 1.008±0.006 0.514±0.010 Mic 0.963±0.002 0.542±0.013 1/2MIC 1.016±0.010 0.574±0.023 1/4MIC 1.029±0.021 0.588±0.011 1/8MIC 1.285±0.037 0.611±0.023 1/16MIC 1.373±0.040 0.647±0.007 1/32MIC 1.570±0.075 0.628±0.031 1/64MIC 1.600±0.011 0.644±0.018 blank control 1.000±0.111 1.000±0.013

Table 5. Dispersion (OD570nm) of Pseudomonas aeruginosa ATCC 27853 biofilm after adding drugs for 24h

concentration TAT C16-TAT 4MIC 0.961±0.067 0.376±0.035 2MIC 1.015±0.119 0.424±0.098 Mic 1.011±0.108 0.547±0.027 1/2MIC 0.976±0.134 0.552±0.067 1/4MIC 0.994±0.186 0.490±0.084 1/8MIC 1.030±0.119 0.497±0.236 1/16MIC 1.044±0.117 0.626±0.181 1/32MIC 1.176±0.111 0.785±0.078 1/64MIC 1.462±0.047 1.023±0.048 blank control 1.000±0.113 1.000±0.134

Table 6. Dispersion (OD570nm) of Pseudomonas aeruginosa ATCC 27853 biofilm after adding medicine 48h

This experiment shows that lipopeptide C12-TAT has a good dispersion effect on Staphylococcus aureus (MRSA) biofilm and C16-TAT on Pseudomonas aeruginosa biofilm, and has an antibacterial biofilm effect.

Embodiment 3, combined application of lipopeptide and antibacterial drug

1. Determination of combined use index (FIC) of lipopeptide and antibacterial drugs against drug-resistant bacteria

Measure respectively the FIC value of the lipopeptide C12-TAT of embodiment 1 and antimicrobial drug combined application to Gram-positive drug-resistant bacterium Staphylococcus aureus ATCC33591 (MRSA), and the C16-TAT of embodiment 1 and antibacterial drug combined application to FIC values of Gram-negative drug-resistant bacteria Pseudomonas aeruginosa ATCC 27853. The method and results are as follows:

Each antimicrobial drug (as drug A) and lipopeptide (as drug B) respectively took their respective 2MIC as the highest concentration, diluted to 8 concentrations with sterile MH broth medium, respectively, along the horizontal axis of the microwell culture plate, On the vertical axis, add 50 μL of MH broth medium containing different concentrations of the two drugs, and then add 100 μL of bacterial solution to make the final bacterial concentration 5×10 ⁴ CFU/well, incubate at 37°C for 18-24 hours, and observe the results. Record the respective MICs when the two drugs are used in combination, and calculate the FIC value according to the following formula.

FIC＝MIC _{A drug combined} /MIC _{A drug alone} + MIC _{B drug combined} /MIC _{B drug alone}

Judgment criteria: FIC≤0.5, synergistic effect; 0.5<FIC≤1, additive effect; 1<FIC≤2, irrelevant effect; FIC>2, antagonistic effect.

The measurement results are as follows:

Table 7. The FIC value of C12-TAT combined with antibacterial drugs against Staphylococcus aureus ATCC33591 (MRSA)

From the results in the above table, it can be seen that C12-TAT combined with vancomycin and boningmycin has an antagonistic effect; C12-TAT combined with clarithromycin has an additive effect; C12-TAT combined with imipenem has an obvious synergistic effect.

Table 8. The FIC value of C16-TAT combined with antibacterial drugs against Pseudomonas aeruginosa ATCC 27853

From the results in the above table, it can be seen that C16-TAT combined with polymyxin and boningmycin has an antagonistic effect; C16-TAT combined with clarithromycin has an additive effect; C16-TAT combined with imipenem has an obvious synergistic effect.

From the above test results, it can be seen that lipopeptides (C12-TAT, C16-TAT) not only have good anti-drug-resistant bacteria activity, but also when specific types of lipopeptides are combined with certain antibacterial drugs to resist drug-resistant bacteria (specifically When C12-TAT+imipenem resists methicillin-resistant Staphylococcus aureus ATCC33591, and C16-TAT+imipenem resists Pseudomonas aeruginosa ATCC27853, it has obvious synergistic effect. It shows that lipopeptide (C12-TAT, C16 -TAT) and imipenem can be used in combination in the antimicrobial field against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

In order to further verify that the combined application of lipopeptides and antibacterial drugs has a better effect on drug-resistant bacteria (C12-TAT+imipenem on MRSA, C16-TAT+imipenem on P. Evaluation of curative effect of combined application on drug-resistant bacterial infection in animals.

2. Evaluation of the curative effect of combined application of lipopeptide and antibacterial drugs on drug-resistant bacterial infection in animals

The curative effect of combined application of lipopeptide and antibacterial drugs on Staphylococcus aureus ATCC33591 (MRSA) or Pseudomonas aeruginosa ATCC 27853 skin infection and lung infection were evaluated respectively (C12-TAT+imipenem, C12-TAT+ clarithromycin For MRSA bacteria; C16-TAT+ imipenem, C16-TAT+ clarithromycin against Pseudomonas aeruginosa ATCC 27853).

2.1 Experimental grouping and dosage

C12-TAT combined application test and C16-TAT combined application test were divided into 1 control group (no drug administration) and 10 drug administration groups respectively. TAT is administered after being dissolved in PBS, and the dosage of C12-TAT is 30 mg/kg body weight; in the C16-TAT (30 mg/kg) group, the dosage of C16-TAT is dissolved in PBS, and the dosage of C16-TAT is 30mg/kg body weight; ②C12-TAT (15mg/kg) group, administer after dissolving C12-TAT with PBS, the dosage of C12-TAT is 15mg/kg body weight; C16-TAT (15mg/kg) group, C16-TAT was dissolved in PBS and administered, and the dose of C16-TAT was 15 mg/kg body weight; ③ imipenem (30 mg/kg) group, imipenem was dissolved in PBS and administered, imipenem The dose of imipenem was 30 mg/kg body weight; ④ imipenem (15 mg/kg) group, dissolved imipenem in PBS and administered, and the dose of imipenem was 15 mg/kg body weight; ⑤ C12 -TAT+imipenem combination (15mg/kg+15mg/kg) group, mix C12-TAT and imipenem at a mass ratio of 1:1, dissolve in PBS and administer, C12-TAT administration The dosage is 15mg/kg body weight, and the dosage of imipenem is 15mg/kg body weight; Mix according to the mass ratio of 1:1, dissolve in PBS and administer, the dosage of C16-TAT is 15mg/kg body weight, and the dosage of imipenem is 15mg/kg body weight; ⑥C12-TAT+imipenem Combined drug (7.5mg/kg+7.5mg/kg) group, mix C12-TAT and imipenem according to the mass ratio of 1:1, dissolve in PBS and administer, the dosage of C12-TAT is 7.5mg /kg body weight, the dose of imipenem is 7.5mg/kg body weight; C16-TAT+imipenem combination (7.5mg/kg+7.5mg/kg) group, C16-TAT and imipenem Mix according to the mass ratio of 1:1, and administer after dissolving with PBS, the dosage of C16-TAT is 7.5mg/kg body weight, and the dosage of imipenem is 7.5mg/kg body weight; ⑦ clarithromycin ( 30mg/kg) group, clarithromycin was dissolved in PBS and administered, and the dosage of clarithromycin was 30mg/kg body weight; ⑧ clarithromycin (15mg/kg) group, clarithromycin was dissolved in PBS and administered The dosage of clarithromycin is 15mg/kg body weight; ⑨C12-TAT+ clarithromycin combination (15mg/kg+15mg/kg) AT and clarithromycin were mixed according to the mass ratio of 1:1, administered after dissolving with PBS, the dosage of C12-TAT was 15 mg/kg body weight, and the dosage of clarithromycin was 15 mg/kg body weight; C16-TAT+ In the clarithromycin combination (15mg/kg+15mg/kg) group, C16-TAT and clarithromycin were mixed according to the mass ratio of 1:1, dissolved in PBS and then administered. The dosage of C16-TAT was 15mg/kg. kg body weight, the dosage of clarithromycin was 15mg/kg body weight; The mass ratio is mixed, administered after dissolving with PBS, the dosage of C12-TAT is 7.5mg/kg body weight, and the dosage of clarithromycin is 7.5mg/kg body weight; C16-TAT+ clarithromycin combination (7.5mg /kg+7.5mg/kg) group, the dosage of C16-TAT is 7.5mg/kg body weight, and the dosage of clarithromycin is 7.5mg/kg body weight.

2.2 Bacterial culture

Pick Staphylococcus aureus ATCC33591 (MRSA) and Pseudomonas aeruginosa ATCC 27853 colonies from the inoculation loop and inoculate them in 5 mL of TSB medium, culture at a constant temperature at 37°C overnight, wash and resuspend with PBS, vortex and mix well, and dilute to 10 with PBS ¹² CFU/mL to obtain a bacterial suspension for later use.

2.3 Construction of mouse infection model

110 male and female ICR mice were randomly divided into 11 groups, with 5 male mice and 5 female mice in each group. The back of each mouse was treated with 2×2cm hair removal and disinfection. Scald the skin on the back of the mouse: put a graduated hollow plastic tube with an inner diameter of 0.6 cm vertically on the back of the mouse, and quickly inject boiling water to the other end of the plastic tube to the 2mL scale , After contacting for 25s, pour out the boiling water. Then inject 50 μL of the prepared Staphylococcus aureus ATCC33591 (MRSA) bacterial suspension or Pseudomonas aeruginosa ATCC27853 bacterial suspension into the back of the scalded mouse, and then inject 50 μL of the prepared gold into the mouse after 24 hours. Staphylococcus aureus ATCC33591 (MRSA) bacterial suspension or Pseudomonas aeruginosa ATCC 27853 bacterial suspension. A mouse model of pneumonia combined with scald-biofilm infection was constructed.

2.4 Administration and treatment

After 24 hours of lung infection treatment, the mice were given 50 μL by nasal inhalation and back injection, respectively, for 3 consecutive days. The dosage of each group was as in 2.1, and the control group was given the same dose of PBS.

2.5 Efficacy evaluation

After the drug treatment, the mice were killed by neck dislocation. Dissection was performed under sterile conditions, local lung tissue and skin tissue were taken out, ground into a homogenate, diluted 10 ⁵ , 10 ⁷ , and 10 ¹⁰ times with PBS, and 100 μL of the diluted solution was spread on a TBS plate. Incubate overnight at a constant temperature of 37°C, and count the colonies in the lung and skin tissues. The result is as follows:

Table 9. Evaluation of curative effect of each administration group on Staphylococcus aureus ATCC33591 (MRSA) pulmonary infection model (n=10)

group Total Bacteria (Log ₁₀ CFU/mL) Control group (no administration) 12.64±1.65 C12-TAT (30mg/kg) 5.75±1.67 C12-TAT (15mg/kg) 8.73±2.66 Imipenem (30mg/kg) 7.13±1.72 Imipenem (15mg/kg) 11.06±1.08 C12-TAT+imipenem combination (15mg/kg+15mg/kg) 3.37±1.72 C12-TAT+imipenem combination (7.5mg/kg+7.5mg/kg) 8.06±1.56 Clarithromycin (30mg/kg) 7.89±1.43 Clarithromycin (15mg/kg) 10.38±2.07 C12-TAT+ clarithromycin combination (15mg/kg+15mg/kg) 4.48±1.56 C12-TAT+ clarithromycin combination (7.5mg/kg+7.5mg/kg) 8.55±1.32

Table 10. Evaluation of curative effect of each administration group on Staphylococcus aureus ATCC33591 (MRSA) skin infection model (n=10)

Table 11. Evaluation of curative effect of each administration group on Pseudomonas aeruginosa ATCC 27853 pulmonary infection model (n=10)

group Total Bacteria (Log ₁₀ CFU/mL) Control group (no administration) 22.89±0.40 C16-TAT (30mg/kg) 19.05±0.93 C16-TAT (15mg/kg) 20.58±0.83 Imipenem (30mg/kg) 18.87±1.62 Imipenem (15mg/kg) 21.21±1.73 C16-TAT+imipenem combination (15mg/kg+15mg/kg) 8.07±4.20 C16-TAT+imipenem combination (7.5mg/kg+7.5mg/kg) 19.04±2.82 Clarithromycin (30mg/kg) 19.15±0.65 Clarithromycin (15mg/kg) 20.37±0.88 C16-TAT+ clarithromycin combination (15mg/kg+15mg/kg) 10.78±1.67 C16-TAT+ clarithromycin combination (7.5mg/kg+7.5mg/kg) 19.63±1.32

Table 12. Evaluation of curative effect of each administration group on Pseudomonas aeruginosa ATCC 27853 skin infection model (n=10)

As can be seen from the above table, compared with the non-administration group, each administration group has therapeutic effects on mice infected with Staphylococcus aureus ATCC33591 (MRSA) and Pseudomonas aeruginosa ATCC 27853, and the total number of bacteria in the lungs and skin decreased significantly. , and the antibacterial effect was drug concentration-dependent. The test results also show that lipopeptides (C12-TAT, C16-TAT) have good antibacterial activity in vivo, which is equivalent to or slightly better than the antibacterial drugs imipenem and clarithromycin at the same dose.

Efficacy of C12-TAT+imipenem on lung infection and skin infection caused by Staphylococcus aureus ATCC33591 (MRSA), C16-TAT+imipenem on lung infection and skin infection caused by Pseudomonas aeruginosa ATCC 27853 , significantly better than the efficacy of the two alone. It shows that the above combination regimen not only has a synergistic effect on drug-resistant bacteria in vitro, but also has a better curative effect than single administration in terms of anti-drug-resistant bacteria infection in animals.

<110> Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences, Peking University

<120> Lipophilic compound conjugates of cell penetrating peptides and their application in antibacterial

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 1

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

Claims (10)

1. Application of a lipopeptide or a pharmaceutically acceptable salt of the lipopeptide or a derivative of the lipopeptide or a multimer of the lipopeptide in the preparation of a product that enhances the bactericidal activity and/or bacteriostatic activity of an antibacterial drug; The lipopeptide is composed of a cell penetrating peptide and a lipophilic compound linked to the amino terminus or carboxyl terminus of the cell penetrating peptide. 2. The application according to claim 1, characterized in that: the cell penetrating peptide is a) or b) polypeptide: a) a polypeptide whose amino acid sequence is SEQ ID No.1; b) a derivative polypeptide obtained by subjecting the polypeptide of a) to substitution and/or deletion and/or addition of 1 to 5 amino acid residues, and the derivative polypeptide has bactericidal activity and/or bacteriostatic activity. 3. The application according to claim 1 or 2, characterized in that: the lipophilic compound is F1, F2 or F3: F1, saturated fatty acid or unsaturated fatty acid; F2, saturated fatty acids containing 8 to 20 carbon atoms; F3, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid. 4. The application according to any one of claims 1 to 3, characterized in that: the derivative of the lipopeptide is the following 1), 2) or 3): 1) Using one or more amino acid residues in the cell penetrating peptide of the lipopeptide with a conformation of D-type amino acid residues, artificially modified amino acid residues and/or rare amino acid residues existing in nature Substitution is carried out to obtain the derived polypeptide: 2) a linker obtained by connecting an amino-terminal protecting group to the amino-terminus of the cell-penetrating peptide of the lipopeptide and/or connecting a carboxy-terminal protecting group to the carboxyl-terminus of the cell-penetrating peptide; 3) A linker obtained by connecting an amino-terminal protecting group to the amino-terminus of the lipopeptide derivative described in 1) and/or a carboxyl-terminal protecting group connected to the carboxyl-terminal of the lipopeptide derivative described in 1). 5. The application according to any one of claims 1 to 4, characterized in that: the multimer of the lipopeptide is a multimer of PM1 or PM2: PM1, a multimer formed by said lipopeptide or a pharmaceutically acceptable salt of said lipopeptide; PM2, a multimer formed by derivatives of said lipopeptide. 6. The application according to any one of claims 1 to 5, characterized in that: the antibacterial drug is a drug containing imipenem or/and clarithromycin. 7. P1, P2 or P3: P1, an antibacterial product, which comprises M1) and M2); said M1) is M11) or/and M12) or/and M13); said M11) is the lipopeptide or lipopeptide described in any one of claims 1-3 The pharmaceutically acceptable salt of the lipopeptide; the M12) is a derivative of the lipopeptide in claim 4; the M13) is a multimer of the lipopeptide in claim 5; The M2) is the antibacterial drug described in claim 1 or 6; P2, antibacterial product, it comprises M1 described in P1); P3, M1 as described in P1). 8. Use of M1) in claim 7 or M1) and M2) in claim 7 in the preparation of P1 or P2 in claim 7. 9. The medicinal compound, characterized in that: the medicinal compound is the M1) described in claim 7. 10. A method for treating or/and preventing pathogenic bacteria from infecting animals, comprising administering M1) or P1 or P2 according to claim 7 to recipient animals to inhibit pathogenic bacteria from infecting animals.