CN107236022B

CN107236022B - Lipophilic compound conjugate of cell penetrating peptide and application thereof in antibiosis

Info

Publication number: CN107236022B
Application number: CN201710418174.3A
Authority: CN
Inventors: 李桂玲; 李馨儒; 于菲菲; 侯续成; 张文茜; 周文凯; 孟艳莎
Original assignee: Peking University; Institute of Medicinal Biotechnology of CAMS
Current assignee: Peking University; Institute of Medicinal Biotechnology of CAMS
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2020-09-29
Anticipated expiration: 2037-06-06
Also published as: CN112107676B; CN107236022A; CN112107676A

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 conjugate of cell penetrating peptide and application thereof in antibiosis

Technical Field

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

Background

Antibiotics are always main medicines for clinical anti-infection treatment, the great success of antibiotics in the last 40 to 70 years reduces the attention and investment on the research and development of antibacterial medicines, the research and development of global antibacterial medicines is slow in the last 40 years, the promotion of medicines with new chemical skeletons, new mechanisms and new targets is very little, and known effective medicines (or derivatives thereof) are continuously and repeatedly used or even abused for many years, so that the medicine effect is reduced, and the medicine resistance is generated.

The problem of increasingly serious bacterial resistance has received widespread attention worldwide. As the drug-resistant strains of various antibacterial drugs continuously appear, the trend of multiple drug resistance is gradually expanded, and the multiple drug resistance has profound influence on clinical medication, so that the problem of drug resistance becomes a new problem in the field of sanitation. In the 60 s of the 20 th century, the number of deaths from infectious diseases worldwide each year was about 700 million, and the age was rising to 2000 million at the beginning of the present era; the number of deaths from sepsis rose by 89%, most of which died from medication difficulties due to super-resistant "superbacteria". WHO established the world day of 2011 topic as "controlling antibiotic resistance: no action is taken today, no drug is available tomorrow, and the severity of bacterial resistance is again documented.

In particular, the increase of multi-drug resistant and pan-drug resistant gram-negative bacteria year by year becomes a difficult point in anti-infective treatment. Gram-negative bacilli (GNBs) are of particular interest because of the ease with which resistance-related genes can be obtained or up-regulated under antibiotic-selective pressure, as compared to gram-positive bacteria. According to the data of the national medical safety network, GNB accounts for more than 30% of nosocomial infectious pathogenic bacteria and is the most common pathogenic bacteria in respiratory-related pneumonia and urinary tract infection. The new delhi metallo beta-lactamase type i (NDM-1) gram-negative bacteria causing extensive drug resistance appeared simultaneously in several regions of the world in 2010 again sounding the alarm clock. These increasing bacterial resistance problems and increasingly complex resistance mechanisms present great difficulties in the treatment of the related diseases and even have severely compromised the health and life of the patients.

Meanwhile, the resistance of bacteria in the form of a biofilm to antibacterial drugs, external environmental stress and the immune system of a host is remarkably enhanced, which is one of the important reasons that bacteria generate drug resistance and infectious diseases are difficult to treat. The bacterial biofilm is a highly organized and systematized bacterial community which is wrapped by extracellular matrix composed of extracellular polysaccharide, protein, nucleic acid and the like secreted by bacteria, mutually adhered and attached to the surface of a certain carrier to grow. The biofilm may be formed by a mixture of one or more bacteria. The biofilm is a growth mode corresponding to planktonic bacteria formed by adapting to living environment in the growth process of bacteria, shows a series of new biological characteristics different from planktonic bacteria on the whole, and has stronger ability of adapting to external environment. The resistance of biofilm bacteria to antibiotics is more than 1000 times that of planktonic bacteria, and the resistance is originated from the multicellular structure of the biofilm. According to the initial statistics of the National Institute of Health (NIH), 80% of bacterial diseases are related to bacterial biofilms, which are widely existed on human respiratory tracts, dental plaques, and artificial devices (such as catheters, artificial valves, stents and the like) which are left in vivo for a short time or a long time. Therefore, the search for a drug-resistant strategy that can target both drug-resistant bacteria and the biofilm of drug-resistant bacteria has become a major breakthrough in the problem of drug resistance against bacteria, particularly gram-negative bacteria.

The use of antibiotics and antibacterial drugs is the most basic and most common means for controlling bacterial biofilm infection, but with the increase of bacterial drug resistance, new active substances and new treatment means different from the traditional antibacterial drugs are also applied to the research field of antibacterial biofilms while effective novel antibacterial drugs are developed.

Cell Penetrating Peptides (CPPs) are a class of short peptides (typically less than 35 amino acid residues) that are capable of entering cells through a variety of cell membranes. This finding was derived from 1988 where the scholars found that Tat, the transactivator of HIV-1, was transduced into cells across the membrane, and subsequently discovered that drosophila transcripts also had similar properties. From these transcription factors, researchers have obtained the shortest peptide sequences required for transduction and found that they can introduce other macromolecules (e.g., proteins, nucleic acids, etc.) into cells. Thereafter, many other CPPs were discovered in succession, and Table 1 lists some of the more representative CPPs.

TABLE 1 representative CPP thereof

The clinically common gram-positive pathogenic bacteria include: staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis), Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus pneumoniae (Streptococcus pneumniae), Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecium (Enterococcus faecalis), Mycobacterium Tuberculosis (MTB), Bacillus subtilis (Bacillus subtilis), Bifidobacterium breve (Bifidobacterium breve) and Bifidobacterium longum (Bifidobacterium longum). Wherein the staphylococcus aureus includes Methicillin-resistant staphylococcus aureus (MRSA) and Methicillin-sensitive staphylococcus aureus (MSSA), the streptococcus pneumoniae includes Penicillin-resistant streptococcus pneumoniae (PRSP) and Penicillin-sensitive streptococcus pneumoniae (PSSP), the enterococcus faecalis includes Vancomycin-resistant enterococcus faecalis (VRE) and Vancomycin-sensitive enterococcus faecalis (VSE-Vancomycin-resistant enterococcus faecalis, VSE), and the enterococcus faecalis includes Vancomycin-resistant enterococcus faecalis (Vancomycin-resistant enterococcus faecalis, VRE), and the enterococcus faecium includes Vancomycin-resistant enterococcus faecalis (Vancomycin-resistant enterococcus faecalis, VRE). Staphylococcus aureus and Staphylococcus epidermidis belong to the genus Staphylococcus (Staphylococcus) of the family Staphylococcus of the order Bacillales (Bacillales) of the class Bacillales (Bacilli) of the phylum Firmicutes (bacillutes) of the bacterial kingdom. Enterococcus faecalis and Enterococcus faecium belong to the genus Enterococcus (Enterococcus) of the family lactococcus (Lactobacillales) of the class Sporobacillus of the phylum firmicutes of the kingdom of bacteria. Streptococcus pyogenes and Streptococcus pneumoniae belong to the genus Streptococcus (Streptococcus) of the order Lactobacillus in the class of Bacillaceae of the phylum firmicutes of the bacterial kingdom. Mycobacterium tuberculosis belongs to the family Mycobacteriaceae (Mycobacterium) Mycobacterium (Mycobacterium) of the order Actinomycetales (Schizomycetes) of the class Schizomycetes (Schizomycetes) of the phylum Mycobacteria of the kingdom of bacteria. Bacillus subtilis belongs to the genus Bacillus (Bacillus) of the family Bacillaceae, order Bacillales, class Bacteromycetes, of the phylum firmicutes of the kingdom of bacteria. Bifidobacterium breve and Bifidobacterium longum belong to the order Bifidobacterium (Bifidobacterium) of the class Actinomycetales (Actinomycetales) actinomycetes (Actinomycetales) of the phylum Actinomycetes (Actinomycetes) of the kingdom of bacteria.

The clinically common gram-negative pathogenic bacteria include: escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Acinetobacter baumannii (Acinetobacter baumannii), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Stenotrophomonas maltophilia (Stenotrophoria), Enterobacter cloacae (Enterobacter cloacae), Citrobacter heterotypii (Citrobacter diversus), Morganella morganii (Morganella morganii), Haemophilus influenzae (Haemophilus flurenzae), Haemophilus parainfluenzae (Haemophilus ainfluezae), and Legionella pneumophila (Legionella pneumophila). Wherein the Escherichia coli belongs to the genus Escherichia (Escherichia) of the family Enterobacteriaceae (Enterobacteriaceae) of the phylum Proteobacteria (Proteobacteria) gamma-Proteobacteria (Gamma Proteobacteria) in the kingdom of bacteria. Klebsiella pneumoniae belongs to the genus Klebsiella (Klebsiella) of the family Enterobacteriaceae of the order Enterobacteriaceae of the class Gamma-Proteobacteria of the phylum Proteobacteria of the kingdom Bacteriae. Acinetobacter baumannii belongs to the family Moraxella (Acinetobacter) of the order Pseudomonas (Pseudomonas) of the order Proteobacteria of the phylum Proteobacteria of the kingdom (Proteobacteria). Pseudomonas aeruginosa belongs to the genus Pseudomonas (Pseudomonas) belonging to the family Pseudomonas of the order Pseudomonas of the phylum Proteobacteria of the kingdom Proteobacteria. Stenotrophomonas maltophilia belongs to the phylum Proteus (Phyllum BX11.Proteobacteria) Gamma Proteobacteria (Gammaproteobacteria) order Xanthomonas (Xanthomonadales) family Xanthomonas (Xanthomonadaceae) genus Stenotrophomonas (Stenotrophoromonas). Enterobacter cloacae belongs to the genus Enterobacteriaceae (Enterobacter) of the order Enterobacteriaceae, the phylum Proteobacteria of the kingdom Bacteromycetes. The heterotypic Citrobacter belongs to the genus Citrobacter (Citrobacter) of the family Enterobacteriaceae, the order Enterobacteriaceae, the phylum Proteobacteria of the Bacteromycetes. Morganella morganii belongs to the genus Morganella of the family Enterobacteriaceae (Morganella) of the order Enterobacteriaceae of the phylum Proteobacteria of the Bacterida of the kingdom fungi. Haemophilus influenzae belongs to the genus Haemophilus (Haemophilus) of the family Pasteurellaceae (Pasteurellales) of the order Pasteurellales (Pasteulales) of the phylum Proteobacteria of the kingdom Bacterida. The haemophilus parainfluenza belongs to the genus haemophilus of the family pasteurellaceae of the order pasteurellaceae of the phylum Proteobacteria of the kingdom of bacteria. Legionella pneumophila is of the genus Legionella (Legionella) of the Legionella family of the order Legionella of the phylum Proteobacteria of the bacterial kingdom.

The clinical common pathogenic fungi comprise: candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Candida parapsilosis (Candida parapsilosis), Aspergillus flavus (Aspergillus flavus), and Aspergillus fumigatus (Aspergillus fumigatus). Among them, Candida albicans, Candida tropicalis and Candida parapsilosis are Candida species of Cryptococcus family, Cryptococcus order, Cryptococcus class, subphylum fungi, of the fungal kingdom. Aspergillus flavus and Aspergillus fumigatus belong to the genus Aspergillus of the family Calycomycetaceae of the order Chimomycetales of the class Deuteromycotina of the kingdom fungi.

Disclosure of Invention

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

In order to solve the above technical problems, the present invention provides a lipophilic compound conjugate of a cell-penetrating peptide (i.e., lipopeptide) or a pharmaceutically acceptable salt thereof.

The lipopeptide provided by the invention is formed by connecting a cell penetrating peptide and a lipophilic compound connected with the amino terminal or the carboxyl terminal of the cell penetrating peptide.

In the above lipopeptide, the cell penetrating peptide is a polypeptide of a) or b):

a) a polypeptide having an amino acid sequence of SEQ ID No. 1;

b) the derivative polypeptide is obtained by substituting and/or deleting and/or adding 1 to 5 amino acid residues on the polypeptide of a), and the derivative polypeptide has bactericidal activity and/or bacteriostatic activity.

In the lipopeptide, the lipophilic compound may be fatty acid or cholesterol, sphinganine or vitamin E.

In the above lipopeptide, the fatty acid may be F1, F2 or F3:

f1, wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid;

f2, wherein the fatty acid is a saturated fatty acid containing 8 to 20 carbon atoms;

f3, wherein 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 having the amino acid sequence of SEQ ID No.1 is a cell penetrating peptide named TAT.

The lipopeptide can be 5 lipopeptides named C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, C8-TAT is formed by connecting TAT and C8 connected with the amino terminal of TAT, C12-TAT is formed by connecting TAT and C12 connected with the amino terminal of TAT, C14-TAT is formed by connecting TAT and C14 connected with the amino terminal of TAT, C16-TAT is formed by connecting TAT and C16 connected with the amino terminal of TAT, and C20-TAT is formed by connecting TAT and C20 connected with the amino terminal of TAT.

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

The pharmaceutical salts of the lipopeptides of the present invention may be formed with cations such as sodium, potassium, aluminum, calcium, lithium, manganese, zinc, and the like, or bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, and the like.

Derivatives of the above lipopeptides also fall within the scope of the present invention.

The derivatives of the lipopeptides provided by the present invention may be 1), 2) or 3) below:

1) a derivative polypeptide obtained by replacing one or more amino acid residues in the cell-penetrating peptide of the lipopeptide with amino acid residues with D-type conformation, artificially modified amino acid residues and/or rare amino acid residues existing in nature;

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

3) a linker obtained by linking an amino terminal protecting group to the amino terminal of the lipopeptide derivative of 1) and/or linking a carboxyl terminal protecting group to the carboxyl terminal of the lipopeptide derivative of 1).

In the lipopeptide derivatives, the D-form amino acid is an amino acid corresponding to the L-form amino acid constituting the protein; the artificially modified amino acid refers to a common L-type amino acid which is modified by methylation, phosphorylation and the like and forms a protein; the rare amino acids existing in nature include unusual amino acids constituting proteins and amino acids not constituting proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, homoserine and the like.

Multimers of the lipopeptides of PM1 or PM2 below are also within the scope of the present invention:

PM1, a multimer formed from the lipopeptide or a pharmaceutically acceptable salt of the lipopeptide;

PM2, multimers formed by derivatives of said lipopeptides.

In order to solve the above technical problems, the present invention provides an application of the lipopeptide or a pharmaceutically acceptable salt of the lipopeptide or a derivative of the lipopeptide or a polymer of the lipopeptide in the preparation of a product (a drug, an antibacterial material or a micellar nanoparticle) for enhancing the bactericidal activity and/or bacteriostatic activity of an antibacterial drug.

In the above application, the antibacterial agent is a drug having bactericidal activity and/or bacteriostatic activity, and includes various chemically synthesized drugs such as antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones, and the like, and also includes some products obtained by culturing microorganisms such as bacteria, actinomycetes, fungi, and the like, or the same or similar substances manufactured by a chemical semi-synthesis method. The antibacterial drug can be specifically a drug containing imipenem or/and clarithromycin.

In the above application, the active ingredient of the antibacterial drug can be imipenem or/and clarithromycin, the active ingredient of the antibacterial drug can further contain other ingredients besides the lipopeptide or the pharmaceutical salt of the lipopeptide or the derivative of the lipopeptide or the polymer of the lipopeptide, and the other active ingredients of the antibacterial drug can be determined by the technicians in the field according to the antibacterial effect.

The following P1 or P2 also belong to the scope of protection of the invention:

p1, antibacterial product comprising M1) and M2); the M1) is M11) or/and M12) or/and M13); said M11) is said lipopeptide or a pharmaceutically acceptable salt of said lipopeptide; said M12) is a derivative of said lipopeptide; said M13) is a multimer of said lipopeptide;

said M2) is said antibacterial agent;

p2, antibacterial product comprising M1 of P1).

The antibacterial product P1 and the antibacterial product P2 can be medicines with bactericidal activity and/or bacteriostatic activity, coatings with bactericidal activity and/or bacteriostatic activity or materials with bactericidal activity and/or bacteriostatic activity. Wherein the coating having bactericidal and/or bacteriostatic activity may be an antibacterial coating for implantation into the surface of an implant of an animal.

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

In the antibacterial product of P1, the active ingredients of the antibacterial product of P1 can be M1) and M2), the active ingredients of the antibacterial product of P1 can also contain other ingredients, and the other active ingredients of the antibacterial product of P1 can be determined by those skilled in the art according to the antibacterial effect.

In the antibacterial product of P2, the active ingredient of the antibacterial product of P2 can be the M1), the active ingredient of the antibacterial product of P2 can also contain other ingredients, and the other active ingredients of the antibacterial product of P2 can be determined by those skilled in the art according to the antibacterial effect.

The antibacterial product of P1 and the antibacterial product of P2 may further comprise pharmaceutically acceptable carriers or excipients. The carrier material includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), sparingly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.).

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

Herein, the bactericidal activity and/or bacteriostatic activity is to have killing and/or inhibiting effects on the following pathogenic bacteria B1 to B23:

b1, gram positive bacteria;

b2, firmicutes bacteria or actinomycetes bacteria;

b3, a bacterium of the class bacillus, a bacterium of the class schizomycete, or a bacterium of the class actinomycete;

b4, a bacterium of the order bacillales, a bacterium of the order lactobacillales, a bacterium of the order actinomycetes or a bacterium of the order bifidobactales;

b5, a bacterium of the family staphylococcaceae, a bacterium of the family enterococcaceae, a bacterium of the family streptococcaceae, a bacterium of the family mycobacteriaceae, a bacterium of the family bacillaceae or a bacterium of the family bifidobacterium;

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, penicillin-resistant Streptococcus pneumoniae, or penicillin-sensitive Streptococcus pneumoniae;

9, vancomycin-, gentamicin-and streptomycin-resistant enterococcus faecalis, vancomycin-and teicoplanin-resistant enterococcus faecium, cephalosporin-and penicillin-resistant streptococcus pneumoniae;

b10, gram-negative bacteria;

b11, proteobacteria or protripes bacteria;

b12, bacteria of the class γ -proteobacteria, bacteria of the class providencia;

b13, a bacterium of the order Enterobacteriales, a bacterium of the order Pseudomonadales, a bacterium of the order Xanthomonas, a bacterium of the order Pasteurellales or a bacterium of the order Legionella;

b14, a bacterium of the enterobacteriaceae family, a bacterium of the moraceae family, a bacterium of the pseudomonadaceae family, a bacterium of the xanthomonadaceae family, a bacterium of the pasteurellaceae family or a bacterium of the legioneaceae family;

b15, an Escherichia bacterium, a Klebsiella bacterium, an Acinetobacter bacterium, a Pseudomonas bacterium, a stenotrophomonas bacterium, an Enterobacter bacterium, a Citrobacter bacterium, an Morganella bacterium, a Haemophilus bacterium, or a Legionella bacterium;

b16, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, stenotrophomonas maltophilia, Enterobacter cloacae, Citrobacter heterotypii, Morganella morganii, Haemophilus influenzae, Haemophilus parainfluenza, or Legionella pneumophila;

b17, fungi;

b18, fungi of the Deuteromycotina;

b19, a fungus of the class sporozoea or deuteromycetae;

b20, fungi of the order cryptococcales or conidiomycetes;

b21, Cryptococcus fungi or Calycopsis fungi;

b22, candida or aspergillus fungi;

b23, Candida albicans, Candida tropicalis, Candida parapsilosis, Aspergillus flavus or Aspergillus fumigatus.

The invention also provides pharmaceutical compounds.

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

The pharmaceutical compound may be a medicament as described above having bactericidal and/or bacteriostatic activity.

The present invention also provides a method for treating or/and preventing infection of an animal with a pathogenic bacterium, comprising administering said M1) or said P1 or said P2 to a recipient animal to inhibit infection of the animal with the pathogenic bacterium.

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

In practical applications, the lipopeptide or its pharmaceutically acceptable salt, the derivative, the polymer, and the pharmaceutical compound of the present invention can be directly administered to patients as drugs, or can be mixed with suitable carriers or excipients to prepare antibacterial drugs for administration to patients, so as to achieve the purpose of treating and/or preventing pathogenic bacteria infection.

Experiments prove that C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, particularly C12-TAT, C14-TAT, C16-TAT and C20-TAT have good inhibition and killing effects on gram-positive bacteria, gram-negative bacteria and fungi, and have broad-spectrum antibacterial effect. Wherein, the C12-TAT has good dispersion effect on methicillin-resistant staphylococcus aureus (MRSA) biomembranes, and the C16-TAT has good dispersion effect on pseudomonas aeruginosa (P. aeruginosa) biomembranes, and has antibacterial biomembrane effect. In the aspect of inhibiting methicillin-resistant staphylococcus aureus, C12-TAT and clarithromycin have additive effects; 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. The C12-TAT and the C16-TAT have good in-vivo antibacterial activity, and the effects of the C12-TAT and the C16-TAT are equivalent to or slightly superior to the activities of antibacterial drugs imipenem and clarithromycin under the same dosage. The curative effect of the C12-TAT and the imipenem on the lung infection and the skin infection caused by methicillin-resistant staphylococcus aureus is obviously better than the curative effect of the C16-TAT and the imipenem on the lung infection and the skin infection caused by pseudomonas aeruginosa when the two are independently administrated. The results show that the combination of C12-TAT and C16-TAT with imipenem not only has synergistic effect on drug-resistant bacteria in vitro, but also is superior to the curative effect of single administration in the aspect of resisting drug-resistant bacteria infection in animals.

Drawings

FIG. 1 is a reaction scheme for the preparation of lipopeptides.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of lipopeptides

This example prepared 6 lipopeptides designated C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT, and C20-TAT, each of the 6 lipopeptides was formed by linking a cell-penetrating peptide (amino acid sequence YGRKKRRQRRR, SEQ ID No.1), designated TAT, to a lipophilic compound attached to the amino terminus of the cell-penetrating peptide.

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

the structural formula of C8-TAT is as follows: C8-YGRKKRRQRRR, wherein C8 is octanoic acid;

the structural formula of C12-TAT is as follows: C12-YGRKKRRQRRR, wherein C12 is lauric acid;

the structural formula of C14-TAT is as follows: C14-YGRKKRRQRRR, wherein C14 is myristic acid;

the structural formula of C16-TAT is as follows: C16-YGRKKRRQRRR, wherein C16 is palmitic acid;

the structural formula of C20-TAT is as follows: 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.

The synthesis method comprises the following steps:

respectively dissolving saturated fatty acids with different carbon chain lengths in acetonitrile, respectively activating with EDC and NHS, and respectively reacting with TAT (at a molar ratio of 1:1) under low temperature and dark conditions, wherein the specific reaction formula is shown in figure 1. In the reaction formula, CH₃(CH₂)_nCOOH represents butyric acid, caprylic acid, lauric acid, myristic acid, palmitic acid or arachidic acid.

The product was subjected to rotary evaporation to remove the solvent, the unreacted raw materials and by-products were removed by column separation, the reaction conversion was measured by HPLC, and the structure of the product was confirmed by NMR, MS and IR. The results show that the C4-TAT, the C8-TAT, the C12-TAT, the C14-TAT, the C16-TAT and the C20-TAT with the structural formula of more than 95% are obtained.

Example 2 antimicrobial action of lipopeptides

1. Determination of Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentration (MIC) of C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT in example 1 is determined by a drug sensitivity test by adopting a dish double dilution method, and the specific test method is as follows: the test bacteria (as shown in table 2) were cultured in MH broth. The drugs (C4-TAT, C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT in example 1) are diluted twice with MH broth culture medium to various required concentrations, an appropriate amount of MH agar culture medium is added into a plate respectively, the MH agar culture medium is melted and then is quantitatively injected into the plate containing the liquid medicine to be mixed evenly, and various microorganisms to be measured are inoculated by a multi-point inoculator (the inoculation amount is 10)⁴CFU/point) is placed in an incubator at 37 ℃ for constant temperature culture for 18h, and the result is observed, and the minimum Concentration of the drug contained in the plate with aseptic growth is the Minimum Inhibitory Concentration (MIC).

The results are as follows:

TABLE 2MIC values of TAT and lipopeptides for each bacterium (. mu.g/mL)

The results show that C8-TAT, C12-TAT, C14-TAT, C16-TAT and C20-TAT, especially C12-TAT, C14-TAT, C16-TAT and C20-TAT have good inhibiting and killing effects on gram-positive bacteria, gram-negative bacteria and fungi, and have broad-spectrum antibacterial effect.

In Table 2, MRSA is methicillin-resistant Staphylococcus aureus, VRE is vancomycin-resistant enterococcus, PSSP is penicillin-sensitive Streptococcus pneumoniae, PRSP is penicillin-resistant Streptococcus pneumoniae; enterococcus faecalis ATCC51575 is resistant to vancomycin, gentamicin and streptomycin, enterococcus faecium ATCC700221 is resistant to vancomycin and teicoplanin, and streptococcus pneumoniae ATCC51915 is resistant to cephalosporin and penicillin.

2. Evaluation of in vitro anti-biofilm Effect

2.1 establishment of in vitro biofilm model

Selecting Staphylococcus aureus ATCC33591(MRSA) or Pseudomonas aeruginosa ATCC27853, placing in 5 mM MH culture solution, performing constant temperature shaking culture at 37 deg.C for 8 hr, and adjusting the concentration of the solution to 1.5 × 10 with turbidimeter⁸CFU/mL, and diluted 100-fold, 2mL of the above-mentioned bacterial solution was aspirated, and the solution was added to a 24-well plate with 8 × 8mm slides and incubated at 37 ℃ to form a mature biofilm.

2.2 quantitative determination of biofilm growth status by Crystal Violet staining

Drug solutions (drugs refer to TAT, C12-TAT and C16-TAT in example 1, and solvents are PBS solutions with pH 7.2) with different concentrations are added into each well respectively, an equal amount of PBS solution with pH 7.2 is added as a blank control, and the wells are incubated at constant temperature of 37 ℃ for 24 hours and 48 hours. After 24 hours and 48 hours, the 24-well plate was gently rinsed with a PBS solution, left to dry. Adding 1% crystal violet solution into the pore plate, staining for 20 minutes, washing the pore plate with PBS solution for 3 times, standing and airing. Adding 95% ethanol into the pore plate, standing for 15 min, eluting crystal violet, measuring absorbance value at 570nm wavelength (lower value represents better splitting effect of biomembrane) with enzyme labeling instrument, and using 95% ethanol as control. And the data were statistically analyzed. The results are shown in tables 3 to 6:

TABLE 3 Dispersion of Staphylococcus aureus ATCC33591(MRSA) biofilm 24h after dosing (OD570nm)

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

Concentration of	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 of Pseudomonas aeruginosa ATCC27853 biofilm after 24h of drug (OD570nm)

Concentration of	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 of Pseudomonas aeruginosa ATCC27853 biofilm after 48h of drug (OD570nm)

The experiment shows that the lipopeptide C12-TAT has good dispersion effect on a staphylococcus aureus (MRSA) biomembrane, and the C16-TAT has good antibacterial biomembrane effect on pseudomonas aeruginosa biomembranes.

Example 3 combination of lipopeptides with antibacterial Agents

1. Combined use index (FIC) determination of drug-resistant bacteria by combined application of lipopeptide and antibacterial agent

The FIC values of the lipopeptide C12-TAT of example 1 in combination with an antibacterial agent against the gram-positive resistant bacterium Staphylococcus aureus ATCC33591(MRSA), and C16-TAT of example 1 in combination with an antibacterial agent against the gram-negative resistant bacterium Pseudomonas aeruginosa ATCC27853 were determined, respectively. The method and the result are as follows:

the antibacterial agent (as drug A) and lipopeptide (as drug B) are diluted to 8 concentrations at 2MIC maximum concentration and sterile MH broth medium multiple ratio, respectively, 50 μ L MH broth medium containing two drugs with different concentrations is added along the horizontal axis and the vertical axis of the micropore culture plate, respectively, and then 100 μ L bacterial liquid is added to make the final bacterial concentration 5 × 10⁴CFU/well, constant temperature culture at 37 ℃ 18The results were observed for 24 h. The MIC of the two drugs was recorded and the FIC value was calculated according to the following formula.

FIC＝MIC_{Combination of medication A}/MIC_{Used singly for Jia Yao}+MIC_{Combination of drugs B}/MIC_{Used singly for treating B}

And (4) judging the standard: FIC is less than or equal to 0.5, and has synergistic effect; FIC is more than 0.5 and less than or equal to 1, and the additive effect is achieved; 1 < FIC <2, irrelevant action; FIC > 2, antagonistic action.

The results of the measurement were as follows:

TABLE 7 FIC values of C12-TAT in combination with antibacterial agents for Staphylococcus aureus ATCC33591(MRSA)

The results in the table show that C12-TAT is combined with vancomycin and boningmycin to have antagonistic action; the C12-TAT has additive effect with clarithromycin; the combination of C12-TAT and imipenem has obvious synergistic effect.

TABLE 8 FIC values of C16-TAT in combination with antibacterial agents for Pseudomonas aeruginosa ATCC27853

The results in the table show that C16-TAT is combined with polymyxin and boningmycin to have antagonistic action; the C16-TAT has additive effect with clarithromycin; the combination of C16-TAT and imipenem has obvious synergistic effect.

From the above test results, it can be seen that the lipopeptides (C12-TAT and C16-TAT) not only have good drug-resistant activity by themselves, but also have significant synergistic effects when a specific kind of lipopeptide is combined with certain antibacterial agents to be applied to drug-resistant bacteria (specifically, C12-TAT + imipenem to methicillin-resistant Staphylococcus aureus ATCC33591 and C16-TAT + imipenem to Pseudomonas aeruginosa ATCC 27853).

In order to further verify that the combined application of the lipopeptide and the antibacterial drug has better effect than single drug application on drug-resistant bacteria (C12-TAT + imipenem to MRSA and C16-TAT + imipenem to P. aeruginosa), the curative effect evaluation of the combined application of the lipopeptide and the antibacterial drug on drug-resistant bacteria infection in animals is carried out.

2. Evaluation of efficacy of lipopeptide and antibacterial drug combined application on drug-resistant bacterial infection in animal body

The efficacy of lipopeptides in combination with antibacterial agents against Staphylococcus aureus ATCC33591(MRSA) or Pseudomonas aeruginosa ATCC27853 skin and lung infections was evaluated (C12-TAT + Imipenem, C12-TAT + clarithromycin against MRSA; C16-TAT + Imipenem, C16-TAT + clarithromycin against Pseudomonas aeruginosa ATCC 27853), respectively.

2.1 Experimental groups and dosages

The C12-TAT combined application test and the C16-TAT combined application test are divided into 1 control group (not administered) and 10 administration groups, wherein the administration groups are respectively as follows: a C12-TAT (30mg/kg) group, wherein C12-TAT is dissolved in PBS and then is administered, and the administration dose of C12-TAT is 30mg/kg body weight; C16-TAT (30mg/kg), wherein C16-TAT is dissolved in PBS and then is administered, and the administration dose of C16-TAT is 30mg/kg body weight; ② a group of C12-TAT (15mg/kg), C12-TAT is dissolved by PBS and then is administrated, the administration dose of C12-TAT is 15mg/kg body weight; C16-TAT (15mg/kg), wherein C16-TAT is dissolved in PBS and then is administered, and the administration dose of C16-TAT is 15mg/kg body weight; ③ group of imipenem (30mg/kg), the imipenem is dissolved in PBS and then is administrated, the dose of the imipenem is 30mg/kg body weight; (15mg/kg) group of imipenem, dissolving imipenem with PBS and then administering, wherein the administration dose of imipenem is 15mg/kg body weight; C12-TAT and imipenem are combined (15mg/kg +15mg/kg) to prepare a group, C12-TAT and imipenem are mixed according to the mass ratio of 1:1, dissolved by PBS and then administered, the administration dose of C12-TAT is 15mg/kg body weight, and the administration dose of imipenem is 15mg/kg body weight; C16-TAT and imipenem are combined to a group (15mg/kg +15mg/kg), C16-TAT and imipenem are mixed according to the mass ratio of 1:1, dissolved by PBS and then administered, the administration dose of C16-TAT is 15mg/kg body weight, and the administration dose of imipenem is 15mg/kg body weight; sixthly, combining C12-TAT and imipenem (7.5mg/kg +7.5mg/kg), mixing C12-TAT and imipenem according to the mass ratio of 1:1, dissolving the mixture by PBS and then administering the mixture, wherein the administration dose of C12-TAT is 7.5mg/kg body weight, and the administration dose of imipenem is 7.5mg/kg body weight; C16-TAT + imipenem combined drug (7.5mg/kg +7.5mg/kg) group, C16-TAT and imipenem are mixed according to the mass ratio of 1:1, dissolved by PBS and then administered, the administration dose of C16-TAT is 7.5mg/kg body weight, and the administration dose of imipenem is 7.5mg/kg body weight; seventhly, a clarithromycin (30mg/kg) group, which is administered after clarithromycin is dissolved in PBS, and the administration dosage of clarithromycin is 30mg/kg body weight; eighty percent (15mg/kg) clarithromycin, which is dissolved in PBS and then administered, wherein the administration dose of clarithromycin is 15mg/kg body weight; ninthly, in the C12-TAT + clarithromycin combination (15mg/kg +15mg/kg) group, C12-TAT and clarithromycin are mixed according to the mass ratio of 1:1 and dissolved in PBS for administration, the administration dose of C12-TAT is 15mg/kg body weight, and the administration dose of clarithromycin is 15mg/kg body weight; C16-TAT + clarithromycin combination (15mg/kg +15mg/kg), C16-TAT and clarithromycin are mixed according to the mass ratio of 1:1, dissolved by PBS and then administered, the administration dosage of C16-TAT is 15mg/kg body weight, and the administration dosage of clarithromycin is 15mg/kg body weight; in a group of combined administration (7.5mg/kg +7.5mg/kg) of C12-TAT and clarithromycin, C12-TAT and clarithromycin are mixed according to a mass ratio of 1:1, and are dissolved in PBS for administration, the administration dose of C12-TAT is 7.5mg/kg body weight, and the administration dose of clarithromycin is 7.5mg/kg body weight; the combination of C16-TAT and clarithromycin (7.5mg/kg +7.5mg/kg) was administered at a dose of 7.5mg/kg body weight for C16-TAT and 7.5mg/kg body weight for clarithromycin.

2.2 bacterial culture

Inoculating loop, selecting Staphylococcus aureus ATCC33591(MRSA) and Pseudomonas aeruginosa ATCC27853, inoculating in 5mL TSB culture medium, culturing at 37 deg.C overnight at constant temperature, washing with PBS for resuspension, vortexing, mixing, diluting with PBS to 10¹²CFU/mL to obtain bacterial suspension for later use.

2.3 mouse infection model construction

The 110 male and female halves of ICR mice were randomly divided into 11 groups of 5 male and 5 female mice each. The back of each mouse was depilated 2X 2cm and sterilized. Scald treatment of the skin of the back of the mouse: a hollow plastic pipe with scales, two ends of which are not provided with plugs and the inner diameter of which is 0.6cm is vertically and tightly attached to the back of a mouse, boiling water is quickly injected into the other end of the plastic pipe to 2mL of scales, and the boiling water is poured out after the plastic pipe is contacted with the scales for 25 s. Then, 50. mu.L of prepared Staphylococcus aureus ATCC33591(MRSA) bacterial suspension or Pseudomonas aeruginosa ATCC27853 bacterial suspension is injected into the back of the scalded mouse, and after 24 hours, 50. mu.L of prepared Staphylococcus aureus ATCC33591(MRSA) bacterial suspension or Pseudomonas aeruginosa ATCC27853 bacterial suspension is injected into the mouse through nasal drip. And (3) constructing a mouse pneumonia combined scald-biofilm infection model.

2.4 treatment with drug administration

After 24 hours of treatment for pulmonary infection, 50 μ L of each drug was administered by nasal drop inhalation and back injection for 3 consecutive days, each group was administered at a dose of 2.1, and the control group was administered at the same dose of PBS.

2.5 evaluation of therapeutic Effect

After the dosing treatment was completed, the mice were sacrificed by cervical dislocation. Dissecting under sterile condition, respectively taking out local lung tissue and skin tissue, grinding into homogenate, and diluting with PBS 10⁵、10⁷、10¹⁰Double, 100. mu.L of the dilution was applied to TBS plates. Incubated overnight at 37 ℃ and subjected to lung and skin tissue colony counting. The results are as follows:

table 9 evaluation of efficacy of each dosing group in staphylococcus aureus ATCC33591(MRSA) lung infection model (n ═ 10)

Group of	Total number of bacteria (Log)₁₀CFU/mL)
		Control group (no drug 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 efficacy of each dosing group on staphylococcus aureus ATCC33591(MRSA) skin infection model (n ═ 10)

Table 11 evaluation of efficacy of each dosing group against pseudomonas aeruginosa ATCC27853 lung infection model (n ═ 10)

Group of	Total number of bacteria (Log)₁₀CFU/mL)
		Control group (no drug 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 efficacy of each administration group on pseudomonas aeruginosa ATCC27853 skin infection model (n 10)

As can be seen from the above table, compared with the non-administration group, each administration group has a therapeutic effect on mice infected with staphylococcus aureus ATCC33591(MRSA) and pseudomonas aeruginosa ATCC27853, the total number of bacteria in lung and skin is significantly reduced, and the antibacterial effect is drug concentration dependent. The test result also shows that the lipopeptide (C12-TAT and C16-TAT) has good in-vivo antibacterial activity, and the effect of the lipopeptide is equivalent to or slightly superior to that of antibacterial drugs of imipenem and clarithromycin under the same dosage.

The curative effect of C12-TAT + imipenem on lung infection and skin infection caused by staphylococcus aureus ATCC33591(MRSA), and the curative effect of C16-TAT + imipenem on lung infection and skin infection caused by pseudomonas aeruginosa ATCC27853 are obviously superior to the curative effect of the two drugs which are independently administrated. The combined medication proposal has synergistic effect on drug-resistant bacteria in vitro and is better than the curative effect of single medication in resisting drug-resistant bacteria infection in animal body.

<110> Beijing university of institute of medical and Biotechnology, national academy of medical sciences

<120> lipophilic compound conjugate of cell penetrating peptide and its application in antibacterium

<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

1. Use of a lipopeptide or a pharmaceutically acceptable salt of said lipopeptide or a multimer of said lipopeptide in the manufacture of a product for enhancing the bactericidal and/or bacteriostatic activity of an antibacterial agent; the lipopeptide is formed by connecting a cell penetrating peptide and a lipophilic compound connected with the amino terminal or the carboxyl terminal of the cell penetrating peptide;

the cell penetrating peptide is a polypeptide with an amino acid sequence of SEQ ID No. 1;

the lipophilic compound is palmitic acid;

the antibacterial drug is imipenem, and the bacteria is pseudomonas aeruginosa.

2. Use according to claim 1, characterized in that: a multimer of said lipopeptide is a multimer formed from said lipopeptide or a pharmaceutically acceptable salt of said lipopeptide.

3. An antimicrobial product characterized by: the antimicrobial product comprises M1) and M2); the M1) is M11 or/and M13); said M11) is the lipopeptide or pharmaceutically acceptable salt of said lipopeptide of claim 1; said M13) is a multimer of the lipopeptide of claim 2;

m2) is the antibacterial agent according to claim 1.