Field of the invention

The present invention relates to the field of biotechnology, biomolecules, and bioinformatics in connection with antimicrobial peptides for inhibition of pathogenic microorganisms in digestive and respiratory tracts in the animal.

Background of the invention

One of the current serious issues that have an impact on humanity is a bacterial infection of gastrointestinal tract (GI tract) and respiratory tract (RI tract) in the animal, which can be transmitted and spread out rapidly as well as leading to an untimely death in case of a delay in medical treatment. Still, a continuous antibiotic therapy increases antibiotic resistance which is likely to rise continuously, and as a result of the resistance, antibiotics lose their ability to kill bacteria. Antibiotic resistance becomes a global issue since it is a fatal emerging infectious disease with no cure. It is a major cause of the spread of antibiotic-resistant bacteria in hospitals and agricultural industries; and also makes an impact on health and economy, causing environmental issues, chemical residues, and food standard problems. In case it happens with the animal in the animal production industry or livestock farm, it would be difficult to deal with a bacterial infection in gastrointestinal tract and respiratory tract in the animal and it would cause widespread damage.

Presently, technology for the treatment of bacterial infection in the animal, especially the infection of gastrointestinal tract and respiratory tract, and for the growth enhancement of the animal in the animal production industry is based on generic antibiotics, e.g. Amoxicillin. Colistin, Doxycycline, Tetracycline, and Tyrosine phosphate. The generic antibiotics are also used for the treatment of human infection. When human directly and indirectly takes antibiotics, there would be an increasing amount of emerging antibiotic-resistant bacteria, which causes more difficulties for the treatment of infected animal and human, especially human who takes antibiotic residues in the animal via consumption. Hence, there is an attempt to find antibiotic alternatives. Each type of antibiotics, in different amount, provides different actions against bacteria; so, in order to overcome the limitations, inventive antibiotic alternatives, in proper amount, must provide broad-spectrum actions against bacteria and provide less side effects. These broad-spectrum antibiotic alternatives currently become desirable so as to prevent antibiotic resistance in the animal in the animal production industry which receives generic antibiotics to kill pathogenic bacteria.

Antimicrobial peptides (AMPs) are one of the antibiotic alternatives used for the medication, prevention, and control of infectious disease. AMPs are basic substances of living beings which have less toxicity, more pathogen specificity, and greater efficiency than antibiotics; cause no antibiotic-resistant bacteria and less side effects; leave no residue in living being or environment; and also provide cell stimulation and control of the immune system. With these properties, AMPs could be a new type of biomolecules providing actions against the growth of pathogenic bacteria, both resistance and non-resistance. They could be new drugs for antibiotic substitution or combination therapy. However, some limitations are found in the current AMPs, e.g. acid intolerance, heat intolerance, cytotoxicity, poor intestinal absorption, and degradation caused by protease in the stomach.

According to the prior art search, there are some application involving the development of antimicrobial peptides for medication, prevention, and substitution of antibiotics. Examples are as follows:

Thai patent application no. 1201005177 discloses short-chain peptides providing actions for the treatment and/or prevention of pathogen infection in gastrointestinal tract in the mammal.

Thai patent application no. 1401005184 discloses short-chain peptides providing actions for the treatment or prevention of inflammation and infection caused by microorganisms.

However, none of the prior art discloses effective broad-spectrum antimicrobial peptides for inhibition of pathogenic bacteria in the animal with the stability in highly-acidic conditions or high temperature. Therefore, the inventor tries to design broad-spectrum antimicrobial peptides that suit for the animal production industry, especially for the treatment of bacterial infection in gastrointestinal tract and respiratory tract in the animal, which helps resolve the problem of drug resistance caused by narrow-spectrum and substantial amounts of antibiotics.

Summary of the invention

The objective of the present invention is to provide broad- spectrum antimicrobial peptides that suit for the animal production industry. In accordance with the principle, biological properties of antimicrobial peptides are subject to peptide size, type of related amino acids, charge, amino acid arrangement, hydrophobicity, and secondary structure. Hence, the inventor applies biomolecular and bioinformatic practice for the modification of peptide size, type of related amino acids, charge, amino acid arrangement, and secondary structure thereof to enhance antibacterial efficiency, especially pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal.

The present invention relates to new artificial antimicrobial peptides providing actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal and cause no toxicity to intestinal cells, lung cells, and red blood cells of the mammal. The antimicrobial peptides are named as GC2K and arranged in helical structure of amino acid sequence of 17 amino acid residues as in the following formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X ₁₁ -X ₁₂ -X ₁₃ -X ₁₄ -X ₁₅ -X ₁₆ -X ₁₇ (I) wherein

X ₁ , X ₅ , X ₉ , X ₁₄ , X ₁₅ , X ₁₇ are Lysine (K),

X ₂ is Tryptophan (W),

X ₃ , X ₇ , X ₁₂ , X ₁₆ are Cysteine (C),

X ₄ is Phenylalanine (F), X ₆ is Valine (V),

X ₈ is Tyrosine (Y),

X ₁₀ is Glycine (G), and X ₁₁ is Isoleucine (I); wherein the antimicrobial peptides become stable when went through the acidity-basicity of pH 2 to 8 for 3 hours and at the temperature of 85°C to 130°C for 10 minutes; wherein the antimicrobial peptides provide actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal; and wherein the antimicrobial peptides cause no toxicity to intestinal cells, lung cells, and red blood cells of the mammal. Besides, the present invention also relates to pharmaceutical components comprising antimicrobial peptides according to the invention and its application for the production of pharmaceutical components providing actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal. Brief description of the drawings

Figure 1 shows an amino acid sequence of antimicrobial peptides for inhibition of pathogenic microorganisms in digestive and respiratory tracts in the animal in helical structure according to the present invention.

Figure 2 shows a three-dimensional structure of antimicrobial peptides for inhibition of pathogenic microorganisms in digestive and respiratory tracts in the animal in helical structure according to the present invention.

Figure 3 shows a graph of a relation between Amoxicillin with the concentration of 0,

1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml and survival rates of 6 species of pathogenic bacteria in gastrointestinal tract, including Salmonella cholerasuis (SC), Salmonella enteritidis (SE), Salmonella typhimiirium (ST), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), and Listeria monocytogenes (LM).

Figure 4 shows a graph of a relation between Colistin with the concentration of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml and survival rates of 6 species of pathogenic bacteria in gastrointestinal tract, including Salmonella cholerasuis (SC), Salmonella enteritidis (SE), Salmonella typhimurium (ST), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), and Listeria monocytogenes (LM).

Figure 5 shows a graph of a relation between Doxycycline with the concentration of 0,

1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml and survival rates of 6 species of pathogenic bacteria in gastrointestinal tract, including Salmonella cholerasuis (SC), Salmonella enteritidis (SE), Salmonella typhimurium (ST), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), and Listeria monocytogenes (LM).

Figure 6 shows a graph of a relation between Tetracycline with the concentration of 0,

1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml and survival rates of 6 species of pathogenic bacteria in gastrointestinal tract, including Salmonella cholerasuis (SC), Salmonella enteritidis (SE), Salmonella typhimurium (ST), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), and Listeria monocytogenes (LM).

Detailed description of the invention The present invention provides artificial antimicrobial peptides using chemical methods wherein the antimicrobial peptides are arranged in helical structure of amino acid sequence of 17 amino acid residues as in the following formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -X ₈ -X ₉ -X ₁₀ -X ₁₁ -X ₁₂ -X ₁₃ -X ₁₄ -X ₁₅ -X ₁₆ -X ₁₇ (I) wherein

X ₁ , X ₅ , X ₉ , X ₁₄ , X ₁₅ , X ₁₇ are Lysine (K),

X ₂ is Tryptophan (W),

X ₃ , X ₇ , X ₁₂ , X ₁₆ are Cysteine (C),

X ₄ is Phenylalanine (F),

X ₆ is Valine (V),

X ₈ is Tyrosine (Y),

X ₁₀ is Glycine (G), and

X ₁₁ is Isoleucine (I); wherein the amino acid sequence of antimicrobial peptides is named as GC2K and arranged in a certain order, KWCFKVCYKGIC YKKCK; wherein the antimicrobial peptides provide actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal; wherein the antimicrobial peptides cause no toxicity to intestinal cells, lung cells, and red blood cells of the mammal; and wherein the antimicrobial peptides become stable when went through the acidity-basicity of pH 2 to 8 for 3 hours and preferably at the acidity of pH 2 for 3 hours.

Besides, the antimicrobial peptides become highly stable when went through the temperature of 85°C to 130°C for 10 minutes; wherein the strong acid tolerance and high stability when went through high temperature will be apt to use in the animal production industry.

In one embodiment, the present invention provides pharmaceutical components comprising antimicrobial peptides according to the invention wherein the pharmaceutical components provide actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal and cause no toxicity to intestinal cells, lung cells, and red blood cells of the mammal treated by said pharmaceutical components.

In another embodiment, the present invention provides the application of antimicrobial peptides according to the invention for the production of pharmaceutical components with actions against the growth of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal.

Methods for the synthesis of antimicrobial peptides according to the invention

Methods for the synthesis of antimicrobial peptides according to the invention are chemical methods, i.e. solid phase peptide synthesis, which can be prepared by the following steps:

A. Blend 9- Fluorenylmethoxycarbonyl (Fmoc) with 9- Fluorenylmethoxycarbonyl (Fmoc) and amino acids as well as 0-benzotriazol-N,N,N-tetramethy-uranium- hexafluorophosphate (HBTU: C ₁₁ H ₁₆ F ₆ N ₅ OP); B. Separate peptides from resin support, resulting in crude peptides;

C. Purify crude peptides using high-performance liquid chromatography (HPLC) to isolate peptide chains; and

D. Find amino acid sequence in the peptide chains using mass spectrometry.

Test GC2K lowest concentration test

A GC2K lowest concentration test according to the invention is prepared by the following steps:

A. Blend 10 μl of peptides at the concentration of 3.3 to 200 μg/ml with 100 μl of bacteria at 10 ⁶ -10 ¹⁰ colony-forming unit/milliliter (CFU/ml) B. Put the mixture derived from A. in a 96-well plate;

C. Place the plate on a 100-RPM shaker and leave for incubation at 37°C; and

D. Follow up on bacterial growth hourly by measuring turbidity (A600) and comparing it with those wells excluding peptides blended by a microplate reader. The microplate reader will then analyze percentage of bacterial growth inhibition at different concentration levels of peptides and antibiotics in comparison with those wells excluding peptides blended by the microplate reader.

GC2K lowest concentration test results

From the GC2K lowest concentration test according to the present invention, GC2K is found to inhibit the growth of 12 species of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal, including Actinobacillus pleuropneumoniae (APP), Avibacterium paragallinarum (APG), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), Listeria monocytogenes (LM), Mycoplasma gallisepticum (MG), Mycoplasma hyopneumoniae (MH), Pasteurella multocida (PM), Salmonella enteritidis (SE), Salmonella typhimurium (ST), Salmonella cholerasuis (SC, and Streptococcus suis (SS), in approximately 7 hours with the volume of only 6.25 to 25 μg/ml wherein the GC2K inhibits 90% of those 12 species. The test results are shown in the following table 1.

Table 1 shows the effective volume of GC2K which inhibits 90% of the growth of 12 species of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal within 7 hours.

The above-mentioned test results of the ability to kill 12 species of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal shows that the GC2K can inclusively kill pathogenic bacteria in both gastrointestinal tract and respiratory tract when comparing to the commonly-used generic antibiotics as shown in the following table 2.

Table 2 Besides, the ability to inclusively kill 12 species of pathogenic bacteria in gastrointestinal tract and respiratory tract in the animal can also be shown by a comparison of killing actions between GC2K and the commonly-used generic antibiotics, i.e. Amoxicillin, Colistin, Doxycycline, and Tetracycline. The killing action results of these antibiotics are shown in figure 3 to figure 10, which show only the actions against the 6 species of pathogenic bacteria, i.e. Salmonella cholerasuis (SC), Salmonella enteritidis (SE), Salmonella typhimurium (ST), Enterohemorrhagic Escherichia coli (EH), Enterotoxogenic Escherichia coli (ET), and Listeria monocytogenes (LM). These 6 species appear in both gastrointestinal tract and respiratory tract, which allow us to see the killing actions of GC2K and 4 types of commonly-used generic antibiotics as shown in the following table 3. Table 3

Test results in the table show that only 25 ppm of GC2K is required to kill all of the 6 species, with broad-spectrum actions. However, such antibiotic as Amoxicillin cannot kill any of these 6 species, while it requires more than 100 ppm of Colistin and Tetracycline and 50 ppm of Doxycycline to do so. Apparently, a large amount of currently-used generic antibiotics is required comparing to the effectiveness of killing actions of GC2K according to the present invention.

GC2K property test GC2K acid-base tolerance test A GC2K property test on acid-base tolerance to GC2K stability wherein a test method can be done by dissolving GC2K in pH 2 to 8 of a buffer solution for 3 hours and then have it tested in comparison with Tetracycline to find the actions against the growth of bacteria.

GC2K acid-base tolerance test results

The above-mentioned test on acid-base tolerance to GC2K stability shows that GC2K highly maintains its stability in the actions against the growth of bacteria at every condition between pH 2 to pH 8. Even solutions of different pH values are applied, GC2K is found to provide effective actions against the growth of bacteria as good as Tetracycline.

GC2K high heat tolerance test A GC2K property test on high heat tolerance to GC2K stability wherein a test method can be done by incubating GC2K solutions at the temperature of 85°C to 130°C for 10 minutes and then have it tested to find the actions against the growth of bacteria.

GC2K high heat tolerance test results

The above-mentioned test on high heat tolerance to GC2K stability shows that GC2K has high heat tolerance of 85°C to 130°C. Favorably, GC2K has the highest heat tolerance of 130°C while still maintaining the actions against the growth of bacteria. The GC2K can also provide effective actions against the growth of bacteria as good as Tetracycline.

Destructive action test on sheep red blood cells

A GC2K property test on destructive actions on sheep red blood cells in comparison with TritonX-100 which destroys 100% of sheep red blood cells can be done by incubating concentrated GC2K solutions of 100, 50, 25, 12.5, 6.25, and 3.125 pg/ml with 1% of concentrated sheep red blood cells at 37°C for 3 hours and measure absorbance of the solutions at 540 nm wavelength, and then compare the absorbance value with those of 1 % of the solutions of concentrated sheep red blood cells, incubated with 1% of concentrated Triton X-100 at 37°C for 3 hours, wherein the 1% of concentrated Triton X-100 is specified to destroy 100% of sheep red blood cells.

Destructive action test results on sheep red blood cells

The GC2K destructive action test on sheep red blood cells in comparison with TritonX- 100 which destroys 100% of sheep red blood cells shows that GC2K at the concentration of 25 μg/ml will not destroy sheep red blood cells, signifying that GC2K according to the present invention is not toxic to sheep red blood cells.

Toxicity test on epithelium cells and lung cells

A GC2K property test on toxicity to epithelium cells and lung cells wherein a test method can be done by incubating GC2K solutions with Hacat cells or MRC5 cells for 24 hours and measure cell viability using MTT assay.

Toxicity test results on epithelium cells and lung cells

The GC2K toxicity test on epithelium cells and lung cells using the above-mentioned method shows that GC2K at the concentration of 25 pg/ml has an influence on Hacat cell viability in a sense that it has low toxicity to the cells, i.e. it approximately destroys 12.9% of the cells, while also affects MRC5 viability since it approximately destroys 3.46% of the cells.

According to the GC2K property test results above, GC2K properties can be concluded as in the following table 4. Table 4

According to the table, GC2K at the concentration of 25 pg/ml has 100% stability to maintain its actions against bacteria when went through acidity-basicity of pH 2 to 8 for 3 hours and when went through the temperature of 85°C to 130°C for 10 minutes. The strong acid and heat tolerance are apt for the application in animal production industry. In addition, GC2K at the concentration of 25 pg/ml do not destroy sheep red blood cells, signifying that the GC2K is not toxic to red blood cells. The GC2K also has low toxicity to cells in gastrointestinal tract and respiratory tract, i.e. only 12.9±1.4% and 3.5±0.0% toxicity to Hacat cells and MRC5 cells accordingly, signifying that the GC2K is not toxic to cells in gastrointestinal tract and respiratory tract. Furthermore, the GC2K is not degraded by such protease as pepsin and trypsin, so enzyme in the stomach has no influence on a loss of peptide properties.

Best mode of the invention

The best method of invention is as described in detailed description of the invention.