WO2017032236A1 - Complex prepared from antimicrobial peptides in combination with polymers, and preparation method and use thereof - Google Patents

Abstract

Provided in the present invention are a complex prepared from antimicrobial peptides in combination with polymers, the preparation method of the complex, and the use of the complex for preparing drugs for preventing or treating diseases or infections caused by bacteria (such as gram-positive bacteria or gram-negative bacteria), fungi or viruses. While the complex of the present invention maintains the antimicrobial peptide activity, the hemolytic toxicity thereof significantly decreases.

Description

Composite of antibacterial peptide and polymer, preparation method and use thereof Technical field

The present application relates to a composite of an antimicrobial peptide and a polymer, and the present application also relates to a method for preparing the composite, and the composite for preparing a prophylactic or therapeutic treatment by bacteria (for example, Gram-positive bacteria or Gram) Use in drugs for diseases or infections caused by negative bacteria, fungi or viruses.

Background technique

Antibacterial peptides are considered to be a new generation of antibiotics because of their broad-spectrum antibacterial activity and their inability to produce drug resistance. These advantages are attributed to their bactericidal mechanism different from current antibiotic drugs: most antimicrobial peptide sequences contain more basic amino acids and are therefore positively charged, easily electrostatically attracted to negatively charged phospholipids on the cell membrane, and then The cationic polypeptide is inserted into the bilayer of the cell membrane to form pores, causing leakage of intracellular fluid, thereby killing the bacteria.

Despite its high antibacterial activity, no antimicrobial peptide drugs have been approved for marketing since the discovery of the first antimicrobial peptide. The biggest obstacle is that the cationic antibacterial peptide has the difficulties encountered in the development of drugs in addition to common peptides. (For example, peptide compounds are easily combined with serum and plasma in the body and degraded by proteolytic enzymes, with short half-life in vivo, poor stability, and antigenicity. Strong and other defects), there are strong side effects such as hemolytic. Therefore, antimicrobial peptides require higher doses when used, which in turn leads to the production of hemolytic toxicity. Therefore, how to obtain stable, safe and effective antimicrobial peptide drugs or preparations has become a challenging and practical research hotspot.

Summary of the invention

The inventors of the present application have made unremitting efforts and a large number of experiments, and surprisingly found that a polyionic complex prepared by electrostatic interaction between an anionic polymer and a cationic antimicrobial peptide can greatly maintain the activity of the antimicrobial peptide. The hemolytic toxicity is lowered, thereby completing the present application.

A first aspect of the present application relates to a complex comprising an antimicrobial peptide and a polymer, wherein the antimicrobial peptide is positively charged and the polymer is negatively charged.

In certain preferred embodiments of the present application, the antimicrobial peptide and polymer are combined by electrostatic interaction.

In certain preferred embodiments of the present application, the antimicrobial peptide is a cationic antimicrobial peptide.

In certain preferred embodiments of the present application, the antimicrobial peptide is selected from the group consisting of a polypeptide, a derivative thereof, a pharmaceutically acceptable salt thereof, and a D-form thereof, or any combination thereof.

In certain preferred embodiments of the present application, the cationic antimicrobial peptide carries 5-20 (eg, 5-15, eg, 5-10,) positive charges.

In certain preferred embodiments of the present application, the cationic antimicrobial peptide is selected from any combination of one or more of Pelican, Omega, HLF1-1, P113, XMP629,

Wherein, the polypeptide sequences of the above antibacterial peptides are respectively:

Pelican SEQ ID No 1: GIGKF LKKAK KFGKA FVKIL KK-NH ₂

Omega South SEQ ID No 2: ILRWP WWPWR RK-NH ₂

LF1-11 SEQ ID No 3: GRRRR RSVQW CA-NH ₂

P113 SEQ ID No 4: AKRHH GYKRK FH-NH 2

XMP629 SEQ ID No 5: KLFR-(3-(1-naphthyl)-A-QAK-(3-(1-naphthyl)-A-NH ₂ .

In certain preferred embodiments of the present application, the cationic antimicrobial peptide is per order.

In certain preferred embodiments of the present application, the cationic antimicrobial peptide is omeganan.

In certain preferred embodiments of the present application, the polymer is biocompatible and biodegradable so that it can be used as a pharmaceutical carrier, adjuvant, excipient, etc., in vivo.

In certain preferred embodiments of the present application, the polymer contains a hydrophilic segment.

In certain preferred embodiments of the present application, the polymer contains a hydrophilic segment and a hydrophobic segment.

In certain preferred embodiments of the present application, the hydrophilic segment is located at the N-terminus of the hydrophobic segment.

In certain preferred embodiments of the present application, the hydrophilic segment comprises acidic ammonia A polymer of a base acid or a derivative thereof.

In certain preferred embodiments of the present application, the hydrophilic segment comprises a copolymer of an acidic amino acid and a hydrophilic amino acid such as serine or threonine or a derivative thereof. For example, a copolymer comprising an acidic amino acid and serine or a derivative thereof; or a copolymer comprising an acidic amino acid and threonine or a derivative thereof.

In certain preferred embodiments of the present application, the N-terminus of the acidic amino acid is further linked to a molecule that initiates polymerization of the amino acid, for example, polyethylene glycol, serine, threonine, C _1-10 alkyl (eg, C _1-6 alkyl) or other small molecular weight compound (molecular weight less than 1000) which initiates polymerization of the amino acid.

In certain preferred embodiments of the present application, the hydrophobic segment comprises a polymer of a hydrophobic amino acid or a derivative thereof.

In certain preferred embodiments of the present application, the acidic amino acid comprises glutamic acid, aspartic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is polyglutamic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is a (glutamate-aspartic acid) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is polyethylene glycol-polyglutamic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is a polyethylene glycol-(glutamate-aspartic acid) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is a (glutamate-aspartate-serine) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is a polyethylene glycol-(glutamate-aspartate-serine) copolymer.

In certain preferred embodiments of the present application, the hydrophobic segment is polyisoleucine.

In certain preferred embodiments of the present application, the hydrophobic segment is polynaphthylalanine.

In certain preferred embodiments of the present application, the polyethylene glycol is PEG or mPEG.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight of from 600 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the polymer is a compound of Formula I:

Wherein R _{1 is} absent or is a molecule capable of initiating polymerization of an amino acid, for example, selected from the group consisting of polyethylene glycol (polyethylene glycol monomethyl ether or polyethylene glycol), serine, threonine, C _1-10 alkyl ( For example, C _1-6 alkyl), or other small molecular weight compound (molecular weight less than 1000) which can initiate polymerization of amino acids;

R _{2 is} selected from -(CH ₂ ) _x COOH and pharmaceutically acceptable salts thereof (eg, sodium salts), wherein x=0-5 (eg, 0, 1, 2, 3, 4, 5);

R ₃ is a residue of a hydrophobic amino acid or a derivative thereof, such as Ile, Leu, Phe, Pro, Val or Trp;

m=5-100, and R ₂ may be the same or different;

n = 0-100, and when n ≥ 2, R ₃ may be the same or different.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight of from 600 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the hydrophobic amino acid derivative is, for example, naphthylalanine, benzyl glutamate or benzyl aspartate, and the like.

In certain preferred embodiments of the present application, the m value is 10-100, such as 15-80, such as 15-51.

In certain preferred embodiments of the present application, the n value is 0 or n is 5-100, such as 5-50, such as 5-30, such as 5-19.

In certain preferred embodiments of the present application, the polymer is selected from the group consisting of a hydrophilic anionic polymer, an amphiphilic diblock anionic polymer, an amphiphilic anionic polymer, or any combination thereof.

In certain preferred embodiments of the present application, the polymer is selected from the group consisting of polyglutamic acid, polyaspartic acid, (glutamic acid-aspartic acid) copolymer, polyethylene glycol-polyglutamic acid , polyethylene glycol-polyaspartic acid, polyethylene glycol-(glutamate-aspartic acid) copolymer, polyglutamic acid- Polyphenylalanine, polyglutamic acid-polyproline, polyglutamic acid-polyleucine, polyglutamic acid-polyisoleucine, polyglutamic acid-polynaphthalene alanine, poly day Aspartic acid-polyphenylalanine, polyaspartic acid-polyproline, polyaspartic acid-polyleucine, polyaspartic acid-polyisoleucine, polyaspartic acid- Polynaphthylalanine, (glutamic acid-aspartic acid) copolymer-polyphenylalanine, (glutamic acid-aspartic acid) copolymer-polyproline, (glutamic acid-associated Copolymer)-polyleucine, (glutamic acid-aspartic acid) copolymer-polyisoleucine, (glutamate-aspartate-serine) copolymer-polyisoleucine , (glutamic acid-aspartic acid) copolymer-polynaphthylalanine, polyethylene glycol-polyglutamic acid-polyleucine, polyethylene glycol-polyglutamic acid-polyproline , polyethylene glycol-polyglutamic acid-polyisoleucine, polyethylene glycol-polyglutamic acid-polyphenylalanine, polyethylene glycol-polyglutamic acid-polynaphthalene, poly Ethylene glycol-polyaspartic acid-polyproline, polyethylene glycol-polyaspartic acid-polyleucine, polyethylene glycol-polyaspartic acid-polyisoleucine, polyethyl b Glycol-polyaspartic acid-poly Alanine, polyethylene glycol-polyaspartic acid-polynaphthylalanine, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyphenylalanine, polyethylene glycol- (glutamic acid-aspartic acid) copolymer-polyproline, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyleucine, polyethylene glycol-(glutamic acid -aspartic acid) copolymer-polyisoleucine, polyethylene glycol-(glutamate-aspartic acid-serine) copolymer-polyisoleucine, polyethylene glycol-(glutamic acid) One of the -aspartic acid) copolymer-polynaphthyl alanine or any combination thereof, wherein the amino acids in the hydrophilic segment may be arranged in any order.

In certain preferred embodiments of the present application, the number of repeating units of amino acids in the hydrophilic segment is from 10 to 100, preferably from 15 to 51.

In certain preferred embodiments of the present application, the number of hydrophobic amino acid repeating units is from 0 to 100, preferably from 5 to 19.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight in the range of from 600 to 20,000, such as from 1,000 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid.

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid-polyisoleucine.

In certain preferred embodiments of the present application, the molar ratio of the polymer to antimicrobial peptide is from 0.02:1 to 50:1, such as from 0.04:1 to 15:1, such as from 0.2:1 to 5:1.

In certain preferred embodiments of the present application, the molar ratio of the polymer to the antimicrobial peptide is 0.04:1, 0.2:1, 0.3:1, 1:1, 3:1, 5:1.

In certain preferred embodiments of the present application, the complex comprises an antimicrobial peptide and a polymer, wherein

The antimicrobial peptide is Pelican or Omega, preferably Pelican;

The polymer is as shown in Formula I,

among them,

R ₁ is polyethylene glycol, and its molecular weight is 600-20000, preferably 2000-5000;

R ₂ is -(CH ₂ ) ₂ COOH or a pharmaceutically acceptable salt thereof (for example, a sodium salt);

R ₃ is a residue of Ile, Leu, Phe, Pro, Val, Trp or a derivative thereof, preferably a residue of Ile;

m is 15-51;

n is 0 or 5-19;

and,

The molar ratio of the polymer to the antimicrobial peptide is from 0.02:1 to 50:1, for example from 0.04:1 to 15:1, for example from 0.2:1 to 5:1.

In certain preferred embodiments of the present application, the residue of Ile is a sec-butyl group.

In certain preferred embodiments of the present application, the complex forms a nanomicelle structure in solution.

In certain preferred embodiments of the present application, the polymer is a triblock polymer, such as a polyethylene glycol-acidic amino acid polymer-hydrophobic polymer segment, the hydrophobic amino acid polymer is located inside the micelle It is an acidic amino acid polymer and an antimicrobial peptide, and the outermost layer is a molecule such as polyethylene glycol which can initiate polymerization of an amino acid.

In certain preferred embodiments of the present application, the positively charged antimicrobial peptide is polymerized A negatively charged group undergoes electrostatic interaction, for example, an amino group in the antimicrobial peptide electrostatically interacts with a carboxyl group of a hydrophilic segment (eg, an acidic amino acid) in the polymer.

A second aspect of the present application is directed to a pharmaceutical composition comprising the complex of the first aspect of the present application, and a pharmaceutically acceptable carrier or excipient.

The third aspect of the present application relates to the complex of the first aspect of the present application for the preparation of a medicament for preventing or treating a disease or infection caused by a bacterium (for example, a Gram-positive or Gram-negative bacterium), a fungus or a virus. use.

In certain preferred embodiments of the invention, the bacteria (eg, Gram-positive or Gram-negative bacteria), fungi, or viruses are susceptible to the antimicrobial peptide.

A fourth aspect of the present invention relates to the use of the complex of the first aspect of the present application for killing or inhibiting the proliferation of bacteria (e.g., Gram-positive or Gram-negative bacteria), fungi or viruses in vitro/in vivo.

The fifth aspect of the present application relates to a method for preparing a composite according to the first aspect of the present application, which comprises the following steps:

Dissolving the antimicrobial peptide and the polymer in water, stirring to mix well, and then separating (removing the unbound material), thereby obtaining the composite, optionally, further comprising the step of lyophilizing after separation;

Preferably, the molar ratio of the polymer to the antimicrobial peptide is from 0.02:1 to 50:1, for example from 0.04:1 to 15:1, for example from 0.2:1 to 5:1.

In certain preferred embodiments of the present application, the antimicrobial peptide is a cationic antimicrobial peptide.

In certain preferred embodiments of the present application, the antimicrobial peptide is selected from the group consisting of a polypeptide, a derivative thereof, a pharmaceutically acceptable salt thereof, and a D-form thereof, or any combination thereof.

For example, the cationic antimicrobial peptide carries 5-20 (eg 5-15, eg 5-10) positive charges, for example selected from Pelican, Omega, hLF1-11, Any combination of any one or more of P113, XMP629.

In certain preferred embodiments of the present application, the cationic antimicrobial peptide is per order.

In certain preferred embodiments of the present application, the cationic antimicrobial peptide is omeganan.

In certain preferred embodiments of the present application, the polymer is an anionic polymer.

In certain preferred embodiments of the present application, the polymer has good biocompatibility and can be used in vivo as a pharmaceutical carrier, adjuvant, excipient, and the like.

In certain preferred embodiments of the present application, the polymer contains a hydrophilic segment.

In certain preferred embodiments of the present application, the polymer contains both a hydrophilic segment and a hydrophobic segment.

In certain preferred embodiments of the present application, the hydrophilic segment is located at the N-terminus of the hydrophobic segment.

In certain preferred embodiments of the present application, the hydrophilic segment comprises a polymer of an acidic amino acid or a derivative thereof.

In certain preferred embodiments of the present application, the hydrophilic segment comprises a copolymer of an acidic amino acid and a hydrophilic amino acid such as serine or threonine or a derivative thereof. For example, a copolymer comprising an acidic amino acid and serine or a derivative thereof, or a copolymer comprising an acidic amino acid and threonine or a derivative thereof.

In certain preferred embodiments of the present application, the N-terminus of the acidic amino acid is further linked to a molecule that initiates polymerization of the amino acid, for example, polyethylene glycol, serine, threonine, C _1-10 alkyl (eg, C _1-6 alkyl) or other small molecular weight compound (molecular weight less than 1000) which initiates polymerization of the amino acid.

In certain preferred embodiments of the present application, the hydrophobic segment comprises a polymer of a hydrophobic amino acid or a derivative thereof.

In certain preferred embodiments of the present application, the acidic amino acid comprises glutamic acid, aspartic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is polyglutamic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is a (glutamate-aspartic acid) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is polyethylene glycol-polyglutamic acid.

In certain preferred embodiments of the present application, the hydrophilic segment is a polyethylene glycol-(glutamate-aspartic acid) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is a (glutamate-aspartate-serine) copolymer.

In certain preferred embodiments of the present application, the hydrophilic segment is a polyethylene glycol-(glutamate-aspartate-serine) copolymer.

In certain preferred embodiments of the present application, the hydrophobic segment is polyisoleucine.

In certain preferred embodiments of the present application, the hydrophobic segment is polynaphthylalanine.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight of from 600 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the polymer is a compound of Formula I:

Wherein R _{1 is} absent or is a molecule capable of initiating polymerization of an amino acid, for example, selected from the group consisting of polyethylene glycol (polyethylene glycol monomethyl ether or polyethylene glycol), serine, threonine, C _1-10 alkyl ( For example, C _1-6 alkyl), or other small molecular weight compound (molecular weight less than 1000) which can initiate polymerization of amino acids;

R ₂ is selected from - (CH _{2) x} COOH and pharmaceutically acceptable salts (e.g. sodium), where x = 0-5 (for example, 0,1,2,3,4,5);

R ₃ is a residue selected from a hydrophobic amino acid or a derivative thereof, and the hydrophobic amino acid is, for example, selected from the group consisting of Ile, Leu, Phe, Pro, Val, Trp;

m=5-100, and R ₂ may be the same or different;

n = 0-100, and when n ≥ 2, R ₃ may be the same or different.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight of from 600 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the hydrophobic amino acid derivative is, for example, naphthylalanine, benzyl glutamate or benzyl aspartate, and the like.

In certain preferred embodiments of the present application, the m value is 10-100, such as 15-80, such as 15-51.

In certain preferred embodiments of the present application, the n value is 0 or n is 5-100, such as 5-50, such as 5-30, such as 5-19.

In certain preferred embodiments of the present application, wherein the polymer is selected from the group consisting of a hydrophilic anionic polymer, an amphiphilic diblock anionic polymer, an amphiphilic anionic polymer, or any combination thereof.

In certain preferred embodiments of the present application, the polymer is selected from the group consisting of polyglutamic acid, polyaspartic acid, (glutamic acid-aspartic acid) copolymer, polyethylene glycol-polyglutamic acid , polyethylene glycol-polyaspartic acid, polyethylene glycol-(glutamate-aspartic acid) copolymer, polyglutamic acid-polyphenylalanine, polyglutamic acid-polyproline , polyglutamic acid-polyleucine, polyglutamic acid-polyisoleucine, polyglutamic acid-polynaphthalene, polyaspartic acid-polyphenylalanine, polyaspartic acid -polyproline, polyaspartic acid-polyleucine, polyaspartic acid-polyisoleucine, polyaspartic acid-polynaphthalene alanine, (glutamate-aspartate) Copolymer-polyphenylalanine, (glutamic acid-aspartic acid) copolymer-polyproline, (glutamic acid-aspartic acid) copolymer-polyleucine, (glutamic acid) -aspartic acid) copolymer-polyisoleucine, (glutamic acid-aspartic acid) copolymer-polynaphthylalanine, (glutamic acid-aspartic acid-serine) copolymer- Polyisoleucine, polyethylene glycol-polyglutamic acid-polyleucine, polyethylene glycol-polyglutamic acid-polyproline, polyethylene glycol-polyglutamic acid-polyisoleucine acid, Ethylene glycol-polyglutamic acid-polyphenylalanine, polyethylene glycol-polyglutamic acid-polynaphthalene alanine, polyethylene glycol-polyaspartic acid-polyproline, polyethylene Alcohol-polyaspartic acid-polyleucine, polyethylene glycol-polyaspartic acid-polyisoleucine, polyethylene glycol-polyaspartic acid-polyphenylalanine, polyethylene Alcohol-polyaspartic acid-polynaphthylalanine, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyphenylalanine, polyethylene glycol-(glutamate-associate Copolymer)-polyproline, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyleucine, polyethylene glycol-(glutamate-aspartic acid) copolymerization -polyisoleucine, polyethylene glycol-(glutamate-aspartic acid-serine) copolymer-polyisoleucine and polyethylene glycol-(glutamate-aspartic acid) copolymerization Or one or any combination of the poly-naphthylalanines, wherein the amino acids in the hydrophilic segment can be arranged in any order.

In certain preferred embodiments of the present application, the amino acid repeats in the hydrophilic segment The number of units is 10-100, preferably 15-51.

In certain preferred embodiments of the present application, the number of hydrophobic amino acid repeating units is from 0 to 100, preferably from 5 to 19.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight in the range of from 600 to 20,000, such as from 1,000 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid.

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid-polyisoleucine.

In certain preferred embodiments of the present application, the molar ratio of the polymer to antimicrobial peptide is from 0.02:1 to 50:1, such as from 0.04:1 to 15:1, such as from 0.2:1 to 5:1.

In certain preferred embodiments of the present application, the molar ratio of the polymer to the antimicrobial peptide is 0.04:1, 0.2:1, 0.3:1, 1:1, 3:1, 5:1.

In certain preferred embodiments of the present application, the complex comprises an antimicrobial peptide and a polymer, wherein

The antimicrobial peptide is Pelican or Omega, preferably Pelican;

The polymer is as shown in Formula I,

among them,

R ₁ is polyethylene glycol, and its molecular weight is 600-20000, preferably 2000-5000;

R ₂ is -(CH ₂ ) ₂ COOH or a pharmaceutically acceptable salt thereof (for example, a sodium salt);

R ₃ is a residue of Ile, Leu, Phe, Pro, Val, Trp or a derivative thereof, preferably a residue of Ile;

m is 15-51;

n is 0 or 5-19;

And,

The molar ratio of the polymer to the antimicrobial peptide is from 0.02:1 to 50:1, for example from 0.04:1 to 15:1, for example from 0.2:1 to 5:1.

In certain preferred embodiments of the present application, the residue of Ile is a sec-butyl group.

The present application also relates to the use of polymers and antimicrobial peptides for the preparation of antimicrobial formulations wherein the polymers are negatively charged.

In certain preferred embodiments of the present application, the polymer is a compound of Formula I:

Wherein R _{1 is} absent or is a molecule capable of initiating polymerization of an amino acid, for example, selected from the group consisting of polyethylene glycol (polyethylene glycol monomethyl ether or polyethylene glycol), serine, threonine, C _1-10 alkyl ( For example, C _1-6 alkyl), or other small molecular weight compound (molecular weight less than 1000) which can initiate polymerization of amino acids;

R _{2 is} selected from -(CH ₂ ) _x COOH and pharmaceutically acceptable salts thereof (eg, sodium salts), wherein x=0-5 (eg, 0, 1, 2, 3, 4, 5);

R ₃ is a residue selected from a hydrophobic amino acid or a derivative thereof, and the hydrophobic amino acid is, for example, selected from the group consisting of Ile, Leu, Phe, Pro, Val, Trp;

m=5-100, and R ₂ may be the same or different;

n = 0-100, and when n ≥ 2, R ₃ may be the same or different.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight of from 600 to 20,000, such as from 1,000 to 15,000, such as from 2,000 to 10,000, such as from 2,000 to 5,000, and the like.

In certain preferred embodiments of the present application, the hydrophobic amino acid derivative is, for example, naphthylalanine, benzyl glutamate or benzyl aspartate, and the like.

In certain preferred embodiments of the present application, the m value is 10-100, such as 15-80, such as 15-51.

In certain preferred embodiments of the present application, the n value is 0 or n is 5-100, such as 5-50, such as 5-30, such as 5-19.

In certain preferred embodiments of the present application, the polymer is selected from the group consisting of a hydrophilic anionic polymer, an amphiphilic diblock anionic polymer, an amphiphilic anionic polymer, or any combination thereof.

In certain preferred embodiments of the present application, the polymer is selected from the group consisting of polyglutamic acid, polyaspartic acid, (glutamic acid-aspartic acid) copolymer, polyethylene glycol-polyglutamic acid , polyethylene glycol-polyaspartic acid, polyethylene glycol-(glutamate-aspartic acid) copolymer, polyglutamic acid-polyphenylalanine, polyglutamic acid-polyproline , polyglutamic acid-polyleucine, polyglutamic acid-polyisoleucine, polyglutamic acid-polynaphthalene, polyaspartic acid-polyphenylalanine, polyaspartic acid -polyproline, polyaspartic acid-polyleucine, polyaspartic acid-polyisoleucine, polyaspartic acid-polynaphthalene alanine, (glutamate-aspartate) Copolymer-polyphenylalanine, (glutamic acid-aspartic acid) copolymer-polyproline, (glutamic acid-aspartic acid) copolymer-polyleucine, (glutamic acid) -aspartic acid) copolymer-polyisoleucine, (glutamic acid-aspartic acid-serine) copolymer-polyisoleucine, (glutamic acid-aspartic acid) copolymer- Polynaphthyl alanine, polyethylene glycol-polyglutamic acid-polyleucine, polyethylene glycol-polyglutamic acid-polyproline, polyethylene glycol-polyglutamic acid-polyisoleucine acid, Ethylene glycol-polyglutamic acid-polyphenylalanine, polyethylene glycol-polyglutamic acid-polynaphthalene alanine, polyethylene glycol-polyaspartic acid-polyproline, polyethylene Alcohol-polyaspartic acid-polyleucine, polyethylene glycol-polyaspartic acid-polyisoleucine, polyethylene glycol-polyaspartic acid-polyphenylalanine, polyethylene Alcohol-polyaspartic acid-polynaphthylalanine, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyphenylalanine, polyethylene glycol-(glutamate-associate Copolymer)-polyproline, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyleucine, polyethylene glycol-(glutamate-aspartic acid) copolymerization -polyisoleucine, polyethylene glycol-(glutamate-aspartic acid-serine) copolymer-polyisoleucine, polyethylene glycol-(glutamate-aspartic acid) copolymerization Or one or any combination of the poly-naphthylalanines, wherein the amino acids in the hydrophilic segment can be arranged in any order.

In certain preferred embodiments of the present application, the number of repeating units of amino acids in the hydrophilic segment is from 10 to 100, preferably from 15 to 51.

In certain preferred embodiments of the present application, the number of hydrophobic amino acid repeating units is from 0 to 50, preferably from 5 to 19.

In certain preferred embodiments of the present application, the polyethylene glycol has a molecular weight range of 600-20000, such as 1000-20000, such as 1000-15000, such as 2000-10000, such as 2000-5000, and the like.

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid

In certain preferred embodiments of the present application, the polymer is polyethylene glycol-polyglutamic acid-polyisoleucine.

The present application also relates to a method of preventing or treating a disease or infection caused by a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus or a virus, comprising administering an effective amount to a subject in need thereof The steps of the complex described herein.

The present application also relates to a complex as described herein for use in the prevention or treatment of a disease or infection caused by a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus or a virus.

The present application also relates to a method of killing or inhibiting the proliferation of bacteria (eg, Gram-positive or Gram-negative bacteria), fungi, or viruses, including to a subject or object (eg, a medical device) in need thereof. The step of administering an effective amount of the complex described herein.

In certain embodiments of the present application, the method is performed in vivo.

In certain embodiments of the present application, the method is performed in vitro.

The present application also relates to the complexes described herein for killing or inhibiting the proliferation of bacteria, such as Gram-positive or Gram-negative bacteria, fungi or viruses.

In certain embodiments of the present application, it is used in an in vivo method. For example, administration to a subject in need thereof.

In certain embodiments of the present application, it is used in an in vitro method. For example, application to an object (such as a medical device) that requires it.

The present application also relates to the use of a complex of the present application for the preparation of a reagent for killing or inhibiting bacteria (such as Gram-positive or Gram-negative bacteria), fungi or The proliferation of the virus.

In certain embodiments of the present application, the agent is for use in an in vivo method. For example, administration to a subject in need thereof.

In certain embodiments of the present application, the agent is for use in an in vitro method. For example, use an object (such as a medical device) that has this need.

In certain preferred embodiments of the present application, the disease caused by a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus or a virus, is sensitive to the antimicrobial peptide.

In certain preferred embodiments of the present application, the bacterium (eg, a Gram-positive or Gram-negative bacterium), fungus, or virus is susceptible to the antimicrobial peptide.

In the present application, the antibacterial peptides refer to a class of small molecule polypeptides having antibacterial activity in various biological innate immune systems, and also include pseudopeptides artificially synthesized using natural antibacterial peptides as templates. According to the source, antimicrobial peptides can be classified into insect antimicrobial peptides, mammalian antimicrobial peptides, plant antimicrobial peptides, microbial antimicrobial peptides, and amphibian antimicrobial peptides. According to the structure, the antimicrobial peptide can be classified into an α-helical antimicrobial peptide, a cysteine-rich antimicrobial peptide, a β-folded antimicrobial peptide, an antimicrobial peptide rich in an amino acid, and an antimicrobial peptide containing a rare amino acid. The antimicrobial peptides described herein include all types of antimicrobial peptides described above, preferably cationic antimicrobial peptides having 5-20 positive charges, such as Pelican, Omega, HLF1-11, P113, XMP629, and the like. Antibacterial peptides have broad-spectrum antibacterial activity and can target Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, tumor cells and the like. The complex of the present application can be used to treat diseases which can be treated by the antimicrobial peptides it contains, and the indications for a particular antimicrobial peptide are known in the art.

In the present application, the cationic antibacterial peptides refer to amphiphilic molecules generally composed of 12-50 amino acid residues produced by plants and animals, which are present in living organisms and are resistant to external microorganisms. Elimination of the function of mutant cells in vivo, including the synthesis of synthetic peptides using natural cationic antimicrobial peptides as templates.

In the present application, the polymer is an anionic polymer, ie a polymer containing a negative charge.

In the present application, the electrostatic effect refers to mutual attraction between opposite charges and mutual repulsion between the same charges.

In the present application, the combination of the antimicrobial peptide and the polymer by electrostatic interaction refers to electrostatic attraction between the carboxyl ion of the anionic polymer and the amino ion of the cationic polypeptide.

In the present application, the polymer of an amino acid is also referred to as a polyamino acid.

In the present application, the hydrophobic amino acid residue refers to a portion remaining after the hydrophobic amino acid removes a group forming a peptide bond, for example, the leucine residue is an isobutyl group, and the phenylalanine residue is a benzyl group. Wait.

In the present application, if not specified, the amino acids are all L-form.

In the present application, the pharmaceutically acceptable salt means (1) an acidic functional group (for example, -COOH, -OH, -SO ₃ H, etc.) present in the complex of the present application and a suitable inorganic or organic cation (base). a salt formed, such as a salt of the complex of the present application with an alkali or alkaline earth metal, an ammonium salt of the complex of the present application, and a salt of the complex of the present application with a nitrogen-containing organic base; and (2) a complex of the present application salts with suitable inorganic or organic anion (acid) form, the present application example, complexes with inorganic acids or organic carboxylic acids salts of basic functional groups present (e.g., -NH _2, etc.).

Thus, when referring to R _{2 in} formula I (which is selected from -(CH ₂ ) _x COOH and pharmaceutically acceptable salts thereof), the pharmaceutically acceptable salts include, but are not limited to, those which form a carboxyl group with R ₂ Alkali metal salt, such as sodium salt, potassium salt, lithium salt, etc.; alkaline earth metal salt, such as calcium salt, magnesium salt, etc.; other metal salts, such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt An inorganic base salt such as an ammonium salt; an organic base salt such as a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, N -methyl glucosamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, proca Salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, tris(hydroxymethyl)aminomethane salt.

When referring to an antimicrobial peptide (which is selected from a polypeptide, a derivative thereof, a pharmaceutically acceptable salt thereof, or a D-isomer thereof), the pharmaceutically acceptable salt includes, but is not limited to, a basic residue that can be associated with the polypeptide a hydrohalide salt formed, such as a hydrofluoride, a hydrochloride, a hydrobromide, a hydroiodide, etc.; a mineral acid salt such as a nitrate, a perchlorate, a sulfate, a phosphate, etc.; a lower alkane Sulfonate, such as a sulfonate, a triflate, an ethanesulfonate, etc.; an aryl sulfonate such as a benzenesulfonate or a p-benzenesulfonate; an organic acid salt such as an acetate, a malate, or a rich Horse salt, succinate, citrate, tartrate, oxalate, maleate, etc.; amino acid salts such as glycinate, trimethylglycine, arginine, ornithine, glutamine Acid salt, aspartate, and the like.

In the present application, the "subject" refers to an animal, in particular a mammal, preferably a human.

In the present application, the "effective amount" means an amount sufficient to obtain or at least partially obtain a desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously. and many more.

DRAWINGS

Figure 1 shows a gel exclusion chromatogram of polymer mPEG ₅₀₀₀ and mPEG ₅₀₀₀ -b-PGlu(OBzl) ₄₉ .

Figure 2 shows the NMR spectra of polyethylene glycol-polyglutamic acid benzyl ester (A) and polyethylene glycol-polyglutamic acid (B).

Figure 3 shows a schematic of the preparation of Complex 1.

Figure 4 shows the particle size distribution (A) and transmission electron micrograph (B) of Composite 1.

Figure 5 shows a gel electrophoresis pattern of Complex 1-6.

Figure 6 shows polyethylene glycol-polyglutamic acid benzyl ester (A), polyethylene glycol-polyglutamic acid benzyl ester-polyisoleucine (B) and polyethylene glycol-polyglutamic acid- Nuclear magnetic spectrum of polyisoleucine (C).

Figure 7 shows an agarose gel electrophoresis pattern of complex 7-12.

Figures 8 to 12 show the results of the hemolytic activity test of persimin and its complexes.

detailed description

The embodiments of the present invention will be described in detail below with reference to the embodiments, but The following examples are intended to illustrate the invention and are not to be considered as limiting the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Example 1 Preparation of anionic polymer mPEG ₅₀₀₀ -b-PGlu ₄₉

mPEG ₅₀₀₀ -NH ₂ (purchased from Kaizheng Bio Co., Ltd.) was dissolved in N,N-dimethylformamide (DMF), and 50-fold mole of glutamic acid-5-benzyl ester-N-carboxyl ring was added. The internal anhydride (Glu(OBzl)-NCA, reference N. Nishiyama, et al, Langmuir 15 (1999) 377-383) was heated to 50 ° C and stirred for 24 hours. The reaction mixture was concentrated, and the concentrate obtained after the solution was poured into diethyl ether, a white precipitate, filtered and dried in vacuo to give a white powder _{mPEG 5000 -b-PGlu (OBzl)} . The molecular weight of the polymer was determined by gel exclusion chromatography (GPC) to be Mn=12214, PDI=1.25. As shown in Fig. 1, the curve of mPEG ₅₀₀₀ -b-PGlu(OBzl) ₄₉ was significantly different from that of mPEG ₅₀₀₀ , indicating that the obtained was obtained. Block polymer. The obtained mPEG _5000- b-PGlu (OBzl) was added to a 0.5 N aqueous sodium hydroxide solution to remove the protecting group, and lyophilized to obtain a white powder. The value of H in the polyethylene glycol repeat unit -CH ₂ CH ₂ O- was compared with the value of H in the benzyl ester moiety benzene ring C ₆ H ₅ - in the polyglutamic acid repeat unit by ¹ H NMR, and m = 49, the product is mPEG ₅₀₀₀ -b-PGlu ₄₉ .

Scheme1 Preparation route of polyethylene glycol monomethyl ether-b-polyglutamic acid

Example 2 Preparation of Composite 1

An aqueous solution of mPEG ₅₀₀₀ -b-PGlu ₄₉ and perylene (synthesized by a microwave polypeptide synthesizer) was mixed at a molar ratio of 1:1, stirred at room temperature for 12 hours, and then dialyzed for 24 hours in a dialysis bag having a molecular weight cut off of 3,500. Lyophilized to obtain Complex 1 (as shown in Figure 3). The particle size of the composite 1 was tested using a laser particle size analyzer (DLS) to be 195.7 ± 5.6 nm, and the particle size distribution was 0.12 ± 0.03 (Table 1). 4 is a transmission electron microscope (TEM) of the composite 1, which shows that the composite 1 is a core-shell structure. It is indicated that the complex 1 is a stable nanomicelle.

Table 1 particle size of the composite and its dispersion index (n = 3).

Example 3 Preparation of Composite 2-4

Percegasan and mPEG ₅₀₀₀ -b-PGlu ₄₉ were mixed at a molar ratio of 1:5, 1:0.2, 1:25, respectively, and composite 2, composite 3, and composite 4 were prepared according to the method of Example 2. .

Example 4 Preparation of anionic polymer mPEG ₅₀₀₀ -b-PGlu

mPEG ₅₀₀₀ -NH _{2 was} dissolved in DMF, followed by addition of different proportions of Glu(OBzl)-NCA, heated to 50 ° C, and stirred for 24 hours. The reaction mixture was then concentrated, and the obtained concentrated crystals crystals crystals crystals The resulting white powder was then added to a 0.5 N aqueous solution of sodium hydroxide to remove the protecting group and lyophilized. The number of repeating units of glutamic acid was determined by ¹ H NMR calculation to be m = 15 and m = 31, i.e., mPEG ₅₀₀₀ -b-PGlu ₁₅ and mPEG ₅₀₀₀ -b-PGlu ₃₁ .

Example 5 Preparation of Composite 5-6

mPEG ₅₀₀₀ -b-PGlu ₁₅ and mPEG ₅₀₀₀ -b-PGlu ₃₁ were separately mixed with perindican at a molar ratio of 1:1, and composite 5 and composite 6 were prepared by the method of Example 2. The particle size and its particle size distribution are shown in Table 1.

Example 6 Gel electrophoresis test of the composite

In order to facilitate the observation of the formation of complexes, the present application uses fluorescently labeled persiganam instead of persiganam, both having the same positive charge number, and fluorescently labeled persiganic has no effect on the preparation of the complex. Add a mixture of complexes 1-6, fluorescently labeled per order, and fluorescently labeled pexigan and polyethylene glycol ₅₀₀₀ to a 1% agarose gel well at a concentration on the gel plate. The two sides were energized at 20 mV, and the direction of movement of the fluorescent substance was observed after 35 minutes. Figure 5 shows fluorescently labeled plastisan. The mixture of fluorescently labeled plastisan and polyethylene glycol ₅₀₀₀ both moved toward the negative electrode and the other complexes moved toward the positive electrode. As the number of anions in the complex increases, the fluorescence intensity increases, indicating that the perindican forms a complex with polyethylene glycol-b-polyglutamic acid, and the stability increases as the number of anions increases.

Example 7 Preparation of anionic polymer mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle

mPEG ₅₀₀₀ -b-PGlu _{49 was} dissolved in DMF and then different ratios of isoleucine-N-carboxycyclolactone (Ile-NCA, reference N. Nishiyama, et al, Langmuir 15 (1999) 377– 383 synthesis), heated to 50 ° C, stirred for 24 hours. The reaction mixture was then concentrated, and the obtained concentrated mixture was evaporated to ethyl ether. The obtained white powder was added to a 0.5 N aqueous sodium hydroxide solution to remove the protecting group, and lyophilized to give a white powder. The value of hydrogen in polyethylene glycol CH ₂ CH ₂ O and the value of hydrogen in CH ₃ CH ₂ CH(CH ₃ ) in polyisoleucine were compared by ¹ H NMR, and n = 5 and n = 13 were calculated. That is, mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₅ and mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₁₃ , Figure 6 is the nuclear magnetic spectrum of the triblock polymer mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₁₃ Figure.

Synthetic route of Scheme 2 triblock polymer mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₁₃

Example 8 Preparation of Composite 7-8

mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₅ and mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₁₃ were respectively mixed with perindiconan in a molar ratio of 1:1, and the composite was prepared by the method of Example 2. 7 and complex 8.

Example 9 Preparation of Composite 9-10

The complex 9 and the composite 10 were prepared by referring to the method of Example 2 by mixing mPEG ₅₀₀₀ -b-PGlu ₄₉ -b-PIle ₁₃ and perindiconan in a molar ratio of 1:3, 3:1, respectively.

Example 10 Preparation of anionic polymer mPEG ₂₀₀₀ -b-PGlu ₅₁ -b-PIle ₁₉

mPEG ₂₀₀₀ -NH _{2 was} dissolved in DMF, then Glu(OBzl)-NCA was added, heated to 50 ° C, and stirred for 24 hours. Then, the reaction mixture was concentrated, and the obtained concentrated solution was poured into diethyl ether, and a white precipitate was precipitated, which was filtered and dried in vacuo to give a white powder. The number of repeating units of glutamic acid m=51 was calculated by ¹ H NMR, i.e., mPEG ₂₀₀₀ -b- PGlu (OBzl) ₅₁ . The obtained mPEG ₂₀₀₀ -b-PGlu(OBzl) _{51 was} dissolved in DMF, and Ile-NCA was added, heated to 50 ° C, and stirred for 24 hours. The reaction mixture was then concentrated, and the obtained concentrated solution was poured into diethyl ether, and the white precipitate was precipitated, filtered, and dried in vacuo to give a white powder. The number of repeating units of isoleucine n=19 was calculated by ¹ H NMR, ie mPEG ₂₀₀₀ - b-PGlu (OBzl) ₅₁ -b-PIle ₁₉ . The obtained mPEG ₂₀₀₀ -b-PGlu(OBzl) ₅₁ -b-PIle _{19 was} added to a 0.5 N aqueous sodium hydroxide solution to remove the protecting group, and lyophilized to obtain a white powder mPEG ₂₀₀₀ -b-PGlu ₅₁ -b-PIle ₁₉ .

Example 11 Preparation of Composite 11

The complex 11 was prepared by referring to the method of Example 2 by mixing mPEG ₂₀₀₀ -b-PGlu ₅₁ -b-PIle ₁₉ and persimin in a molar ratio of 1:1.

Example 12 Preparation of Composite 12

The complex 12 was prepared by referring to the method of Example 2 by mixing mPEG ₂₀₀₀ -b-PGlu ₅₁ -b-PIle ₁₉ and persimin in a molar ratio of 1:5.

Example 13 Gel electrophoresis test of the complex

In order to facilitate the observation of the formation of complexes, the present application uses fluorescently labeled persiganam instead of persiganam, both having the same positive charge number, and fluorescently labeled persiganic has no effect on the preparation of the complex. Adding Complex 7-12, Fluorescently Labeled Persimin, and a mixture of fluorescently labeled Pexigan and Polyethylene Glycol ₅₀₀₀ to a 1% agarose gel well at a concentration on the gel plate The two sides were energized at 20 mV, and the direction of movement of the fluorescent substance was observed after 35 minutes. Figure 7 shows fluorescently labeled pessimin. The mixture of fluorescently labeled plastisan and polyethylene glycol ₅₀₀₀ both moved toward the negative electrode and the other complexes moved toward the positive electrode. As the number of anions increases, the fluorescence intensity increases, indicating that perindananan forms a complex with polyethylene glycol-b-polyglutamic acid-b-polyisoleucine, and as the number of anions increases Increased stability.

Example 14 Determination of antibacterial activity of a compound containing per order

The experimental strains (Gram-positive bacteria: Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria: Escherichia coli) were inoculated separately into nutrient broth and cultured at 37 ° C for 18-24 hours, and the tested OD ₄₉₀ value was 0.5 or so. Immediately before use diluted 1:10 ⁵ for each species, the bacterial culture tube 12 taken and numbered. Pexigan or its complexes were formulated into a stock solution at a concentration of 1 mg/mL (both in terms of perindican), diluted in dilution, each concentration was repeated 3 times, and 100 μL of bacterial solution was added. Gently shake gently, incubate at 37 ° C for 18-24 hours and observe. After the culture, the culture tube was clarified, and after clarification, it was still clarified, and the tube was considered to be aseptically grown; the turbid state showed growth of bacteria. The culture tube with the lowest concentration of Persimin was found from each tube that was aseptically grown, and the concentration of the tube was the minimum inhibitory concentration (MIC) for inhibiting bacterial growth. The antibacterial results are shown in Table 2. Although the anionic polymer had no antibacterial activity, the complex showed significant antibacterial activity against the three common strains and was comparable to the positive control Pelican antibacterial activity.

Table 2 Minimum inhibitory concentration (MIC) of plastisan and its complexes with polymers

Example 15 Determination of hemolytic activity of a perindopram-containing complex

The human red blood cells are formulated into a red blood cell suspension (diluted 10 times), and the perindopril or complex is formulated into a stock solution having a concentration of 1 mg/mL (both in terms of perindican). Dilute, repeat three times for each sample, shake the shaker after adding the red blood cell suspension. After centrifugation, the supernatant was taken to determine the OD value at a wavelength of 414 nm in a microplate reader, and the red blood cells were 0 in the PBS solution (negative control), and the red blood cells were 100% hemolyzed in the Triton X-100 (positive control). . The percentage of hemolysis is calculated by:

Wherein A is the OD value at 414 nm.

Figures 8 - 12 show a comparison of the hemolytic activity of perindiconan and complexes, and the results show that each complex significantly reduces the hemolytic activity on human red blood cells.

Table 3 compares the therapeutic index (HC ₁₀ /MIC) of per order and its complexes [Chen Y, Mant CT, Farmer SW, Hancock REW, Vasil ML, Hodges RS. J Biol Chem 2005; 280: 12316–29 [Zhu WL, Nan YH, Hahm KS, Shin SY.J Biochem Mol Biol 2007; 40:1090–4.], the therapeutic index is an important indicator for evaluating drug safety, and the larger the value, the safer the drug is. Table 3 shows that the therapeutic index of the complex is increased, and the higher the number of negative charges, the higher the therapeutic index.

Table 3 Comparison of therapeutic indices (HC ₁₀ /MIC) of per order and its complexes

Example 16 Preparation of Composite 13

An aqueous solution of mPEG ₅₀₀₀ -b-PGlu ₁₅ and omeganan (synthesized by a microwave polypeptide synthesizer) was mixed at a molar ratio of 1:1, and a composite 13 was obtained by the method of Example 2.

Example 17 Preparation of Composite 14

An aqueous solution of mPEG ₅₀₀₀ -b-PGlu ₃₁ and omegagan (synthesized by a microwave polypeptide synthesizer) was mixed at a molar ratio of 1:1, and a composite 14 was prepared by the method of Example 2.

Example 18 Preparation of Composite 15

An aqueous solution of mPEG ₅₀₀₀ -b-PGlu ₄₉ and omegagan (synthesized by a microwave polypeptide synthesizer) was mixed at a molar ratio of 1:1, and a composite 15 was prepared by the method of Example 2.

Example 19 Determination of Antibacterial Activity of Complex Containing Omegagan

The MIC of each complex was measured by the method of Example 14, and as a result, as shown in Table 4, the complex showed significant bacteriostatic activity against three common strains, and was comparable to the positive control Pessimin bacteriostatic activity.

Table 4 Minimum inhibitory concentration (MIC) of perindananan and its complex with polymers

Example 20 Complex mouse acute toxicity test

18 to 22 g of healthy balb/c male mice were used as experimental subjects. Mice were randomized by body weight according to the principle of randomization. 8-10 mice per group were set. Different doses of the drug were administered by intraperitoneal injection in a single administration (Pessamican group, 25, 30, 35, 40, 45 and 50 mg/kg; complex 1 group, 600, 800, 1000, 1100, 1200) , 1300 and 1400 mg/kg, of which about 20% of per order was added. After the administration, the death was observed daily for a week. Data analysis was performed using OriginPro 8 software to calculate the LD _{50 of} each of the positive drug and the test drug.

Pessimin group: Mice usually die within half an hour to 48 hours after administration, depending on the dose administered, and then survive substantially. After intraperitoneal injection, the mice in the high-dose group immediately showed extreme discomfort, contracture, vertical hair, eyeballs, limbs, and tails all blue until death. If the mouse can recover from the blue to the original redness, it will not die. Animals in the low dose group performed normally.

Table 5 Dosage grouping and mortality of mice in the Pexiganan group:

The data was analyzed using OriginPro 8 software and the median lethal dose LD50 = 38.86 mg/kg was calculated.

Compound 1 group:

Acute toxicity: same as positive control.

Table 6 Group dose and death of compound 1 group:

The data was analyzed using OriginPro 8 software and the median lethal dose LD50 = 986.6 mg/kg was calculated. According to the actual usage of Pelican, the LD50 is increased by 5 times.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (23)

1. a complex comprising an antimicrobial peptide and a polymer, wherein the antimicrobial peptide is positively charged and the polymer is negatively charged;

   Preferably, the antimicrobial peptide is a cationic antimicrobial peptide, for example, the cationic antimicrobial peptide has a positive charge of 5-20;

   Preferably, the antimicrobial peptide is selected from the group consisting of a polypeptide, a derivative thereof, a pharmaceutically acceptable salt thereof, and a D-form thereof, or any combination thereof.
2. The complex of claim 1 wherein said cationic antimicrobial peptide is selected from the group consisting of Pelican, Omega, HLF1-11, P113, XMP629, or any combination thereof.
3. The composite according to claim 1 or 2, wherein said polymer contains a hydrophilic segment;

   Preferably, the polymer contains both a hydrophilic segment and a hydrophobic segment;

   Preferably, the hydrophilic segment is located at the N-terminus of the hydrophobic segment;

   Preferably, the hydrophilic segment comprises a polymer of an acidic amino acid or a derivative thereof;

   Preferably, the hydrophilic segment comprises a copolymer of an acidic amino acid and a hydrophilic amino acid such as serine or threonine or a derivative thereof; for example, a copolymer comprising an acidic amino acid and serine or a derivative thereof, or a copolymer of an acidic amino acid and threonine or a derivative thereof; optionally, the N-terminus of the acidic amino acid is further linked to a molecule capable of initiating polymerization of an amino acid, such as polyethylene glycol, serine, threonine, C _{1- a 10} alkyl group (for example, a C _1-6 alkyl group) or another small molecular weight compound (having a molecular weight of less than 1000) capable of initiating polymerization of an amino acid;

   Preferably, the hydrophobic segment comprises a polymer of a hydrophobic amino acid or a derivative thereof.
4. The complex of any one of claims 1 to 3, wherein the polymer is a compound of formula I:

   

   Wherein R _{1 is} absent or is a molecule capable of initiating polymerization of an amino acid, for example, selected from the group consisting of polyethylene glycol (polyethylene glycol or polyethylene glycol monomethyl ether), serine, threonine, C _1-10 alkyl ( For example, C _1-6 alkyl), or other small molecular weight compound (molecular weight less than 1000) capable of initiating polymerization of an amino acid;

   R _{2 is} selected from -(CH ₂ ) _x COOH and pharmaceutically acceptable salts thereof (eg, sodium salts), wherein x=0-5 (eg, 0, 1, 2, 3, 4, 5);

   R _{3 is} selected from a hydrophobic amino acid residue or a derivative thereof, and the hydrophobic amino acid is selected from the group consisting of Ile, Leu, Phe, Pro, Val, and Trp;

   m=5-100, and R ₂ may be the same or different;

   n = 0-100, and when n ≥ 2, R ₂ may be the same or different.
5. The complex according to any one of claims 1 to 4, wherein the polymer is selected from the group consisting of polyglutamic acid, polyaspartic acid, (glutamic acid-aspartic acid) copolymer, polyethylene glycol-poly Valley Acid, polyethylene glycol-polyaspartic acid, polyethylene glycol-(glutamate-aspartic acid) copolymer, polyglutamic acid-polyphenylalanine, polyglutamic acid-polyfluorene Acid, polyglutamic acid-polyleucine, polyglutamic acid-polyisoleucine, polyglutamic acid-polynaphthalene, polyaspartic acid-polyphenylalanine, polyaspartic -polyproline, polyaspartic acid-polyleucine, polyaspartic acid-polyisoleucine, polyaspartic acid-polynaphthalene alanine, (glutamate-aspartate Copolymer)-polyphenylalanine, (glutamic acid-aspartic acid) copolymer-polyproline, (glutamic acid-aspartic acid) copolymer-polyleucine, (Valley -aspartic acid) copolymer-polyisoleucine, (glutamic acid-aspartic acid) copolymer-polynaphthylalanine, (glutamic acid-aspartic acid-serine) copolymerization -polyisoleucine, polyethylene glycol-polyglutamic acid-polyleucine, polyethylene glycol-polyglutamic acid-polyproline, polyethylene glycol-polyglutamic acid-polyiso Leucine, Ethylene glycol-polyglutamic acid-polyphenylalanine, polyethylene glycol-polyglutamic acid-polynaphthalene alanine, polyethylene glycol-polyaspartic acid-polyproline, polyethylene Alcohol-polyaspartic acid-polyleucine, polyethylene glycol-polyaspartic acid-polyisoleucine, polyethylene glycol-polyaspartic acid-polyphenylalanine, polyethylene Alcohol-polyaspartic acid-polynaphthylalanine, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyphenylalanine, polyethylene glycol-(glutamate-associate Copolymer)-polyproline, polyethylene glycol-(glutamate-aspartic acid) copolymer-polyleucine, polyethylene glycol-(glutamate-aspartic acid) copolymerization -polyisoleucine, polyethylene glycol-(glutamate-aspartate Acid-serine) copolymer - one of polyisoleucine and polyethylene glycol-(glutamate-aspartic acid) copolymer - polynaphthyl alanine or any combination thereof, wherein the hydrophilic chain The amino acids in the segments can be arranged in any order.
6. The complex according to any one of claims 1 to 5, wherein the molar ratio of the polymer to the antimicrobial peptide is from 0.02:1 to 50:1, for example from 0.04:1 to 15:1, for example from 0.2:1 to 5:1. .
7. A pharmaceutical composition comprising the complex of any of claims 1-6, and a pharmaceutically acceptable carrier or excipient.
8. Use of the complex according to any one of claims 1 to 6 for the manufacture of a medicament for preventing or treating a disease or infection caused by a bacterium such as a Gram-positive or Gram-negative bacterium, a fungus or a virus.
9. The use of claim 8, wherein the disease or infection is sensitive to the antimicrobial peptide.
10. Use of a complex according to any of claims 1-6 for killing or inhibiting the proliferation of bacteria, such as Gram-positive or Gram-negative bacteria, fungi or viruses, in vitro/in vivo.
11. A method of preparing a composite according to any one of claims 1 to 6, comprising the steps of:

    Dissolving the antimicrobial peptide and the polymer in water, stirring to mix well, and separating, to obtain the composite, optionally, further comprising the step of lyophilizing after separation;

    Preferably, wherein the molar ratio of the polymer to the antimicrobial peptide is from 0.02:1 to 50:1, for example from 0.04:1 to 15:1, for example from 0.2:1 to 5:1.
12. A method of preventing or treating a disease or infection caused by a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus or a virus, comprising administering to a subject in need thereof an effective amount of claim 1 The step of the complex of any of -6.
13. The complex according to any one of claims 1 to 6 for use in the prevention or treatment of a disease or infection caused by a bacterium such as a Gram-positive or Gram-negative bacterium, a fungus or a virus.
14. A method of killing or inhibiting the proliferation of a bacterium (eg, a Gram-positive or Gram-negative bacterium), fungus, or virus, comprising administering an effective amount to a subject or object (eg, a medical device) in need thereof The step of the composite of any of claims 1-6.
15. The method of claim 14 which is carried out in vivo.
16. The method of claim 14 which is carried out in vitro.
17. The complex of any of claims 1-6 for use in killing or inhibiting the proliferation of bacteria, such as Gram-positive or Gram-negative bacteria, fungi or viruses.
18. The complex of claim 17 for use in an in vivo method.
19. The complex of claim 17 for use in an in vitro method.
20. Use of a complex according to any of claims 1-6 for the preparation of a reagent for killing or inhibiting the proliferation of a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus or a virus .
21. The use of claim 20, wherein the agent is for use in an in vivo method.
22. The use of claim 20, wherein the agent is for use in an in vitro method.
23. The method, complex or use of any one of claims 14 to 22, wherein the bacterium (e.g., Gram-positive or Gram-negative), fungus or virus is sensitive to the antimicrobial peptide.

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