WO2011120359A1

WO2011120359A1 - Low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof

Info

Publication number: WO2011120359A1
Application number: PCT/CN2011/070970
Authority: WO
Inventors: Jya-Wei Cheng; Kuo-Ch Huang; His-Tsu Cheng; Hui-yuan YU
Original assignee: Rise Technology Co., Ltd.
Priority date: 2010-03-29
Filing date: 2011-02-14
Publication date: 2011-10-06
Also published as: CN102206250B; JP6124139B2; DE112011100556B4; JP2013523661A; CN102206250A; DE112011100556T5

Abstract

Disclosed is an antimicrobial peptide having an amino acid sequence of formula presented as (P₁)_M(A₁X₁X₂)_N(P₂)_X, wherein Pi is selected from the group consisting of basic amino acids including Arg and Lys; A₁ is selected from the group consisting of aromatic amino acids including Trp, Phe and Ala; X₁ is selected from the group consisting of basic amino acids or nonpolar amino acids, including Arg, Lys, VaI, Leu, Ala and Ile; X₂ is selected from the group consisting of basic amino acids or nonpolar amino acids, including Arg, Lys, VaI, Leu, Ala and Ile; P₂ is selected from the group consisting of basic amino acids including Arg and Lys; and the numbers of M and X are respectively 0∼2; when N>2, A₁ is Ala and the Ala residues are less than N-2.

Description

Low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof

FIELD OF THE INVENTION

[0001] The present invention relates to an antimicrobial peptide, and particularly relates to a low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof.

BACKGROUND OF THE INVENTION

[0002] The emergence of bacterial strains that are resistant to conventional antibiotics has prompted a search for new therapeutic agents, including antimicrobial peptides of animal origin. Antimicrobial peptides have been recognized as playing an important role in the innate host defense mechanisms of most living organisms including those of plants, insects, amphibians and mammals, and are known to possess potent antibiotic activity against bacteria, fungi, and certain viruses. The antimicrobial peptides readily partition into phospholipid bilayers with greater than 95% of the peptides binding to lipid to compromise membrane integrity. In bacteria, antimicrobial peptides are able to cause small, transient increases in conductance in planar lipid bilayers, thereby partially depolarizing the cytoplasmic membrane potential gradient.

[0003] The protective function of antimicrobial peptides in innate host defense mechanisms has been demonstrated in Drosophila, where reduced expression of such peptides dramatically decreases survival rates after microbial challenge. In mammals, a similar function is suggested by defective bacterial killing in the lungs of cystic fibrosis patients and in small mice.

[0004] The antimicrobial peptides found in mammals may be classified into the cysteine-rich defensins (a- and β-defensin) and various groups within the cathelicidin family. Based on the amino acid composition and structure, the cathelicidin family may be classified into three groups. The first group includes the amphipathic a-helical peptides such as LL-37, CRAMP, SMAP-29, PMAP-37, BMAP-27, and BMAP-28. The second group contains the Arg/Pro-rich or Trp-rich peptides including Bac5, Bac7, PR-39, and indolicidin. The third group includes Cys-containing peptides such as protegrins. Cathelicidin families contain a highly-conserved signal sequence and proregion known as the cathelin domain and a variable antimicrobial sequence in the C-terminal domain. Many cathelicidins contain a characteristic elastase cleavage site between the anionic cathelin domain and the cationic C-terminal peptide domain. Proteolytic processing at this site has been observed in bovine and porcine neutrophils and is required for microbicidal activity. Although these antimicrobial peptides have a broad spectrum of activity against many microbial organisms, they may have different hemolytic activities for erythrocytes, so that their pharmaceutical potential is restricted. Therefore, the low hemolytic antimicrobial peptides are disclosed in the present invention, they have not only the outstanding antimicrobial activities but also have low hemolytic activities as well.

[0005] As antimicrobial peptides are low molecular mass molecules of less than 5 kDa possessing broad-spectrum activity and constituting an important part of the host defense against microbial infections, they provide a starting point for designing low molecular mass antibiotic compounds. Furthermore, they are known to have a propensity to fold into amphipathic structures with clusters of hydrophobic and charge regions, a feature contributing to their membranolytic activity. Despite these antimicrobial peptides having a broad spectrum of activity against many microbial organisms, they may have different hemolytic activities for erythrocytes, so that their pharmaceutical potential is restricted. SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to provide a low hemolytic antimicrobial peptide, composition and use thereof.

[0007] In one embodiment of the present invention, the antimicrobial peptide of the present invention has an amino acid sequence of formula presented as (PI)M(AIXIX₂)N(P2)X, wherein Pi is selected from the group consisting of basic amino acids including Arg and Lys; Ai is selected from the group consisting of aromatic amino acids including Trp, Phe and Ala; Xi is selected from the group consisting of basic amino acids or nonpolar amino acids, including Arg, Lys, Val, Leu, Ala and He; X₂ is selected from the group consisting of basic amino acids or nonpolar amino acids, including Arg, Lys, Val, Leu, Ala and He; P₂ is selected from the group consisting of basic amino acids including Arg and Lys; and the numbers of M and X are respectively 0-2; when N>2, Ai is Ala and the Ala residues are less than N-2.

[0008] By modifications of primary and secondary structures, the peptides can be obtained and some of their important features can be also analyzed, so as to improve the activity or toxicity of nature antimicrobial peptides. The antimicrobial peptides of the present invention are novel and tryptophan-rich peptides. The peptides have outstanding antimicrobial and low hemolytic activity so that they are suitable for manufacture of antibiotics, and can be used to broadly resist gram-positive bacteria, gram-negative bacteria, protozoa, fungi or Human immunodeficiency virus (HIV).

[0009] In another embodiment of the present invention, the antimicrobial peptide is selected from the group consisting of SEQ ID NO: 1-7, has linear or cyclic conformation, and can be further modified by acetylation, amidation, formylation, hydroxylation, lipid modification, methylaiton or phosphorylaiton. [00010] The peptides of the present invention may improve the activity or toxicity of nature antimicrobial peptides. In the future, they can be used to manufacture antibiotics, pharmaceutical composition or for other clinical antimicrobial uses. Through their outstanding antimicrobial effect and low hemolytic activity, these peptides may broadly resist microorganisms such as gram-positive bacteria, gram-negative bacteria, protozoa, fungi or viruses.

[00011] In yet another embodiment of the present invention, the antimicrobial peptide of the present invention and pharmaceutically acceptable carrier can be used to manufacture pharmaceutical composition as antimicrobial agent. The carrier is an excipient, diluent, thickening agent, bulking agent, binder, disintegrating agent, lubricant, oil-based/ non-oil-based agent, surfactant, suspending agent, gelling agent, adjuvant, preservative agent, anti-oxidant, stabilizing agent, coloring agent, or flavoring agent. The dosage form of the pharmaceutical composition is an embedding, dip, infusion, patch, powder, tablet, injection, suspension, external aqueous solution, drop, liniment, inhalant, embrocation, paste, lotion, cream, ointment, or gel. The composition can be administered to mammals by means of oral, subcutaneous, injective or inhalation administration.

[00012] The embodiments of the present invention are further described through below detailed examples and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[00013] Figure 1 shows hemolytic activities of Pem-2252, Pem-2254 and melittin.

DETAILED DESCRIPTION

[00014] Embodiment 1: Design, synthesis, purification and characterization of peptides

[00015] The antimicrobial peptides of the present invention are listed as table 1, the amino acid residues are presented by 3 -letter abbreviation.

Table 1 name amino acid sequence ID NO

Pern- 1001 C Lys Phe Lys Arg Trp Leu Ala SEQIDNO: 1

Pern- 1001 L Lys Phe Lys Arg Trp Leu Ala SEQIDNO: 1

Pern- 1002 C Lys Phe Arg Ala Trp Val Arg SEQIDNO: 2

Pern- 1002 L Lys Phe Arg Ala Trp Val Arg SEQIDNO: 2

Pern- 1003 C Lys Trp Lys He Trp Leu Lys SEQIDNO: 3

Pern- 1003 L Lys Trp Lys He Trp Leu Lys SEQIDNO: 3

Pem-2251 C Lys Lys Trp Arg Ala Trp Leu Lys Trp Leu Ala Lys Lys SEQIDNO: 4

Pem-2251 L Lys Lys Trp Arg Ala Trp Leu Lys Trp Leu Ala Lys Lys SEQIDNO: 4

Pem-2252 C Lys Lys Trp Arg Lys Trp Leu Arg Ala He Ala Lys Lys SEQIDNO: 5

Pem-2252 L Lys Lys Trp Arg Lys Trp Leu Arg Ala He Ala Lys Lys SEQIDNO: 5

Pem-2253 C Lys Lys Phe Arg Arg Phe Val Arg Phe He Ala Lys Lys SEQIDNO: 6

Pem-2253 L Lys Lys Phe Arg Arg Phe Val Arg Phe He Ala Lys Lys SEQIDNO: 6

Pem-2254 C Lys Lys Trp Arg Arg Trp Leu Lys Trp Leu Ala Lys Lys SEQIDNO: 7

Pem-2254 L Lys Lys Trp Arg Arg Trp Leu Lys Trp Leu Ala Lys Lys SEQIDNO: 7

Note: C and L denote cyclic or linear topology

[00016] All of the cyclic and linear peptides herein were synthesized by solid-phase peptide synthesis using the standard Fmoc (N-(9-fluoroenyl)methoxycarbonyl) protocol manually on PAL resin (5-(4-Fmoc-aminomethyl -3,5-dimethoxyphenoxy - valeric acid - MBHA). Fmoc protective groups of the resin were removed by 20% piperidine/DMF for 1-1.5 hr and checked by ninhydrin test. 95% TFA was added and mixed for 1-1.5 hr to obtain the crude peptides. The crude peptides were then analyzed and purified by reverse phase high pressure liquid chromatography (RP-HPLC) using a Vydac C18 re versed-phase column. The mobile phase for elution was a mixture of acetonitrile and deionized H₂0 mixed in different ratios using gradient elution. The wavelength for detection was set at 225 nm and 280nm, and the flow rate for elution was 4 ml/min. The major peptide products were characterized by fast atom bombard mass spectrophotometry to determine the molecular weight of each peptide. The purity of each peptide was analyzed by RP-HPLC.

[00017] Embodiment 2: Determination of peptide activity in vitro

[00018] The in vitro antimicrobial activities of antimicrobial agents were tested using minimum inhibition concentration (MIC) tests. The MIC value is the lowest concentration of peptide at which the visible growth of test organisms was inhibited and reduced. The test strains herein were E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 29213).

[00019] Overnight cultures of the test organisms were diluted to produce an inoculum containing approximately 10⁵ colonies in Meuller-Hinton broth (MHB). Peptide solution with different concentrations was added to diluted culture of the test organisms. After 18 hours of incubation at 37°C , the results were assayed for turbidity as an indicator of cell growth. MIC values for the peptides were measured three times at different time points. The mean MIC values are shown in Table 2. According to the results, Pem-2251 L and Pem-2254 L showed the better antimicrobial activity against E. coli, Pseudomonas aeruginosa and Staphylococcus aureus. In particular, the MIC values of Pem-2254 L were 1.565, 1.565 and 3.125 ^g/ml) for E. coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. On the other hand, modifications for primary or secondary structures without influencing their activity, such as acetylation, amidation, formylation, hydroxylation, lipid modification, methylaiton or phosphorylaiton, were performed herein. The peptide activity in vitro was then tested and also exhibited their antimicrobial activity.

[00020] Table 2

MIC ^g/ml)

peptide name E. coli P. aeruginosa S. aureus

(ATCC 25922) (ATCC 27853) (ATCC 29213)

Pern- 1001 C >50 >50 >50

Pern- 1001 L 50 50 50

Pern- 1002 C >50 >50 >50

Pern- 1003 L 25 12.5 12.5

Pem-2251 C 25 25 50

Pem-2251 L 1.565 6.25 12.5

Pem-2252 C >50 >50 >50

Pem-2252 L 25 25 25

Pem-2253 C 50 50 50

Pem-2253 L 12.5 12.5 12.5

Pem-2254 C 25 25 50

Pem-2254 L 1.565 1.565 3.125 [00021] For Pem-2251 L and Pem-2254 L peptides, having higher antimicrobial activity as shown in table 2, the MIC values were then tested in IX PBS against various strains such as Bacillus substilis, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus pumilus, Bacillus cereus, Pseudomonas aeruginosa and E. coli, respectively. The MIC values against various strains are shown in table 3, Pem-2251 L and Pem-2254 L have great microbial activity as well.

[00022] Table 3

MIC ^g/ml)

strains

Pem-2251 L Pem-2254 L

B. substilis 3.125 3.125

S. epidermidis 3.125 1.565

S. aureus 3.125 1.565

B. pumilus 6.25 3.125

B. cereus 6.25 6.25

P. aeruginosa 6.25 3.125

E. coli 3.125 1.565

[00023] Embodiment 3: Membrane permeabilization assays

[00024] The outer membrane permeabilization activity of the peptide variants was determined by the 1-N-phenylnaphthylamine (KPN) uptake assay, using intact cells of E. coli. NPN exhibits weak fluorescence in an aqueous environment but exhibits strong fluorescence in a hydrophobic environment. Since NPN is hydrophobic, it provides a direct measurement of the degree of outer membrane permeability. E. coli take up little or no NPN in a general condition. In the presence of permeabilizer compounds (EDTA, polymyxin B, Neomycin, or antimicrobial peptides), NPN partitioned into the bacterial outer membrane resulted in an increase in fluorescence. Fluorescence would vary with the concentration of peptide. The steps of the embodiment were described as follow. One ml of overnight culture was used to inoculate 50 ml of media and incubated at 37°C with shaking. The culture was permitted to grow to an OD₆oo of 0.4 to 0.6, cells were spun down at 3500 rpm for 10 minutes, washed, and re-suspended in buffer to an OD₆oo of 0.5. The OD₆oo was recorded, 1 ml of cells (OD₆oo = 0.5) was added to the cuvette and measured after 2 - 5 seconds. 20 μΐ NPN 0.5 mM, shaken to mix, was added and measured after 2 - 5 seconds. 10 ul antibiotic 100X desired final concentration was added, shaken to mix, and measured until the maximal value was reached within 1 to 5 minutes. The concentration of peptide leading to 50% of the maximum increase in NPN uptake was recorded as the P50. As results shown in table 4, all of the peptides were capable of interacting with membrane.

[00025] Table 4: Ability to permeablize and promote NPN uptake across outer membrane of E. coli peptide P₅₀ ^g ml)

Pern- 1001 L 6.25

Pem-2251 L 6.25

[00026] Embodiment 4: Hemolytic assay

[00027] Melittin, Pem-2252 L and Pem-2254 L were tested for hemolysis against human red blood cells (hRBC). Melittin is a peptide extracted from bees and possesses high hemolytic activity for erythrocytes and used as an experiment control herein. The hRBCs with EDTA were rinsed 3 times with PBS (800 xg, 10 min) and re-suspended in PBS. The hRBCs were diluted into 10% with phosphate-buffered saline and placed 50μ1 into each eppendorf. The peptides dissolved in PBS were then added to 50μ1 of 10% solution of hRBCs and incubated for an hour at 37°C (final hRBC concentration, 5% v/v). The samples were centrifuged at 800g for 10 min at OD₄₀5. Various concentrations of peptides were incubated with pretreated hRBC and the percentage of hemolysis determined ( Percentage lysis ' Zero hemolysis (blank) and 100% hemolysis were determined in PBS buffer and 1% Triton X-100 ). As the results shown in table 5 and fig. 1, Pem-2252 was less hemolytic against hRBC than other antimicrobial peptides. At 5 μg/ml, 50 μg/ml and 400 μg/ml, its lysis percentages were 0.45 %, 1.52 %and 16.35 %, respectively.

Table 5 lysis percentage (%) peptide name 5μg/ml 50μg/ml 400μg/ml

Melittin 50 100 100

Pem-2252 L 3.12 9.82 37.66

Pem-2254 L 045 L52 16.35

[00028] With reference to the above embodiments, the peptides of the present invention may improve the activity or toxicity of nature antimicrobial peptides. In the future, they can be used to manufacture antibiotics, pharmaceutical composition or for other clinical antimicrobial uses. Through their outstanding antimicrobial effect and low hemolytic activity, these peptides may broadly resist microorganisms such as gram-positive bacteria, gram-negative bacteria, protozoa, fungi or viruses. [00029] The antimicrobial peptide of the present invention and pharmaceutically acceptable carrier can be used to manufacture pharmaceutical composition as antimicrobial agent. The carrier is an excipient, diluent, thickening agent, bulking agent, binder, disintegrating agent, lubricant, oil-based/ non-oil-based agent, surfactant, suspending agent, gelling agent, adjuvant, preservative agent, anti-oxidant, stabilizing agent, coloring agent, or flavoring agent. The dosage form of the pharmaceutical composition is an embedding, dip, infusion, patch, powder, tablet, injection, suspension, external aqueous solution, drop, liniment, inhalant, embrocation, paste, lotion, cream, ointment, or gel. The composition can be administered to mammals by means of oral, subcutaneous, injective or inhalation administration.

[00030] Although the present invention has been described with reference to the preferred embodiments thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.

Claims

1. An antimicrobial peptide, having an amino acid sequence of formula presented as follow:

(P₁)_M(A₁X₁X₂)_N(P₂)_X wherein Pi is selected from the group consisting of basic amino acids; wherein Ai is selected from the group consisting of aromatic amino acids or alanine; wherein Xi is selected from the group consisting of basic amino acids or nonpolar amino acids; wherein X₂ is selected from the group consisting of basic amino acids or nonpolar amino acids; wherein P₂ is selected from the group consisting of basic amino acids; and the numbers of M and X are respectively 0~2, the number of N is 2~4.

2. The antimicrobial peptide as claimed in claim 1, wherein Ai is alanine and the numbers of the Ai are less than N-2, while N> 2.

3. The antimicrobial peptide as claimed in claim 1 being selected from the group consisting of SEQ ID NO: 1-7.

4. The antimicrobial peptide as claimed in claim 1 being a linear chain peptide.

5. The antimicrobial peptide as claimed in claim 1 being a cyclic peptide.

6. The antimicrobial peptide as claimed in claim 1 further being modified by acetylation, amidation, formylation, hydroxylation, lipid modification, methylaiton or phosphorylaiton.

7. A use of antimicrobial peptide as antimicrobial agent, comprising administrating the antimicrobial peptide as claimed in claim 1 to a subject.

8. The use as claimed in claim 7, wherein the antimicrobial peptide has low hemolytic activity for the erythrocytes of the subject.

9. The use as claimed in claim 7 being against gram-positive bacteria, gram-negative bacteria, protozoa, fungi or viruses.

10. The use as claimed in claim 7, wherein the subject is a mammal.

11. The use as claimed in claim 7, wherein the subject is administrated with the antimicrobial peptide by means of oral, subcutaneous, injective or inhalation administration.

12. A pharmaceutical composition as antimicrobial agent, comprising the antimicrobial peptide as claimed in claim 1 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition as claimed in claim 12, wherein the carrier is an excipient, diluent, thickening agent, bulking agent, binder, disintegrating agent, lubricant, oil-based/ non-oil-based agent, surfactant, suspending agent, gelling agent, adjuvant, preservative agent, anti-oxidant, stabilizing agent, coloring agent, or flavoring agent.

14. The pharmaceutical composition as claimed in claim 12, wherein the dosage form of the pharmaceutical composition is an embedding, dip, infusion, patch, powder, tablet, injection, suspension, external aqueous solution, drop, liniment, inhalant, embrocation, paste, lotion, cream, ointment, or gel.