RU2448725C2 - Method of increasing bactericidal activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medical and veterinary microbiology and deals with method of increasing bactericidal activity. Gram-positive and Gram-negative bacteria are subjected to impact by synthetic peptides which have structure LYS GLY PRO LEU THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO or THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO, with molecular weight 1794 or 1397 dalton respectively, dissolved in physiological solution in amount 10-30 mg of synthetic peptides per 1 ml of physiological solution.

EFFECT: invention provides novel antibacterial peptides which possess high bactericidal activity not only in buffer solutions but also in physiological solution.

1 tbl, 2 ex

Description

The invention relates to medicine, namely to allergology and immunology, surgery, microbiology, therapy, pharmacology, clinical pharmacology, veterinary medicine and can be used as a method of antibacterial exposure to gram-positive and gram-negative bacteria.

Despite a fairly wide range of antibacterial drugs, the search and development of new drugs with antibacterial activity remains relevant to this day.

Today, one of the most promising areas is the development of antibacterial agents based on endogenous neutrophilic peptides. Due to the pronounced antimicrobial properties and small size of the molecules, these peptides are considered as potential agents for the treatment of human infectious diseases.

As a prototype used known recombinant peptides with antibacterial activity: HNP 1-3 and LL37.

- LL37 - cathelicidin family of antimicrobial peptides of human neutrophils.

- HNP (human neutrophils peptides), containing 30 amino acid residues. The secondary structure of HNP is represented by a β-folded antiparallel structure stabilized by three intramolecular disulfide bridges between cysteine residues. Budikhina A.S., Pinegin B.V. α-Defensins - antimicrobial peptides of neutrophils: properties and functions. Immunology. No. 5. Volume 29, p. 317-320; 2008. Immunology. No. 5. Volume 29, p. 317-320; 2008 year

The undoubted advantages of these peptides over antibiotics are a wider spectrum of antibacterial action, functional activity at low concentrations, almost complete absence of the possibility of formation of resistance of pathogens to antimicrobial peptides, the possibility of synthesis of analogues of natural peptides with altered biological properties.

However, a significant drawback of these peptides is the lack of bactericidal activity when dissolved in physiological saline, which is a significant obstacle to the prospects of their use as a medicine.

The objective of the proposed method is the use of a synthetic peptide agent with high bactericidal activity against gram-positive and gram-negative bacteria and retaining its antibacterial properties both in physiological saline and in buffer media.

The problem is solved in that in the method of increasing bactericidal activity, which consists in influencing gram-positive and gram-negative bacteria with peptides used in a pharmaceutical basis, according to the invention, synthetic peptides having the structure LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS are used as peptides GLN HIS CYS PRO and THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO, with a molecular weight of 1794 and 1397 daltons, respectively, saline solution based on 10 ÷ 30 mg of synthetic peptides, 1 ml phi saline solution.

Due to the fact that synthetic peptides having the structure LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO and THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO, s with a molecular weight of 1794 and 1397 daltons, respectively, a physiological solution of 10–30 mg of synthetic peptides, 1 ml of physiological saline is used as a medicinal base, antibacterial activity against gram-positive and gram-negative bacteria is increased, which retains its properties and buffered medium.

The inventive method of increasing the bactericidal activity of the body has a novelty in comparison with the prototype, which differs in essential features, since a new chemical compound with a known structure is obtained having the properties of both a substance having antibacterial activity both when dissolved in physiological solution and in special buffer media, which he had not previously been described anywhere, while the degree of severity of antibacterial activity in buffer media compared with the known synthetic ptidami in this matter does not change, then there are all the signs of achieving the desired result.

The applicant is not aware of technical solutions that have the indicated distinguishing features, which together ensure the achievement of a given result, so he believes that the claimed method meets the criterion of "inventive step".

The inventive method can be widely used in allergology and immunology, surgery, microbiology, therapy, pharmacology, clinical pharmacology.

Evaluation of the bactericidal activity of the proposed method is as follows.

The bactericidal activity of fractions of cell supernatants was evaluated according to the generally accepted methodology "Determination of the sensitivity of microbes to antibiotics by diffusion into agar using paper discs." In sterile Petri dishes, 20 ml of 2% standard meat-peptone agar was poured. Sowing a test culture of microorganisms was carried out in a conventional manner. The hospital strain of St.aureus 155, E. Coli 2290 and Canda albicans was seeded onto a dried surface of the medium with a continuous lawn using a sterile cotton swab dipped in a 2-billion suspension of microbial culture. Disks impregnated with the studied substances were laid on the surface of the seeded medium by immersing the disks in the composition until completely saturated.

Zones of inhibition of growth of microorganisms were taken into account after 18 hours, measuring their diameter in millimeters with a ruler.

Evaluation of the bactericidal activity of the tested substances in relation to the hospital strains of St.aureus 155, E. Coli 2290 and Canda albicans are presented in table 1. The obtained results demonstrate the presence of bactericidal activity of the following samples: 1 peak MNC, intercid, peptide Zp-1 (LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO), Zp-2 peptide (THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO), LL37 (prototype), HNP (prototype).

It was found that commercial bactericidal endogenous peptides (BEP) LL37 (prototype) and HNP (prototype), dissolved in a buffer solution at a dose of 10 mg / ml (20.1 ± 1.9 and 22), have pronounced bactericidal activity against St. Aureus 155 2 ± 2.15 mm diameter of the zone of inhibition of growth of the microorganism, respectively). At the same dose, they exhibit the highest bactericidal activity against E. Coli 2290 (23.2 ± 1.7 and 24.7 ± 2.15 mm in diameter of the zone of inhibition of microorganism growth, respectively). The activity obtained by us peptide ZP-2, consisting of 12 amino acids, has similar activity. His bactericidal activity against St. Aureus 155 and E. Coli 2290 did not significantly differ from the well-known commercial BEP and amounted to 22.3 ± 2.16 and 19.6 ± 1.6 mm, respectively, of the diameter of the zone of inhibition of growth of microorganisms. The only difference was that a similar bactericidal activity was manifested not only in the buffer solution, but also when dissolved in physiological saline. When dissolving bactericidal endogenous peptides (BEP) LL37 and HNP in physiological saline, the latter do not exhibit bactericidal activity, which is also seen from table 1.

Table 1 Characterization of the bactericidal activity of the investigated substances No. Research tools Concentration [mg / ml] n The diameter of the suppression zone, mm St.aureus 155 E. coli 2290 Canda albicans 8 Peptide ZP-1 in saline 3 10 0 0 0 10 10 10.5 ± 1.24 0 0 thirty 10 10.6 ± 0.6 0 0 9 Peptide ZP-2 in saline 3 10 0 0 0 10 10 12.2 ± 1.1 7.9 ± 0.46 0 thirty 10 22.6 ± 2.23 20.1 ± 1.8 0 10 ZP-1 Peptide in Buffer Solution 3 10 0 0 0 10 10 11.5 ± 1.09 0 0 thirty 10 9.6 ± 0.8 0 0 12 ZP-2 Peptide in Buffer Solution 3 10 0 0 0 10 10 11.7 ± 1.2 7.3 ± 0.5 0 thirty 10 22.3 ± 2.16 19.6 ± 1.6 0 13 LL37 (prototype) in saline 3 10 0 0 0 10 10 0 0 0 thirty 10 0 0 0 fourteen HNP (prototype) in saline 3 10 0 0 0 10 10 0 0 0 thirty 10 0 0 0 fifteen LL37 (prototype) in a buffer solution 3 10 0 0 0 10 10 20.1 ± 1.9 23.2 ± 1.7 0 thirty 10 19.4 ± 2.16 19.4 ± 1.7 0 16 HNP (prototype) in buffer solution 3 10 17.3 ± 1.2 13.3 ± 1.1 0 10 10 22.2 ± 2.15 24.7 ± 2.15 0 thirty 10 20.4 ± 1.8 20.4 ± 1.8 0

With respect to Canda albicans, none of the studied peptides showed bactericidal activity, including commercial BEP (LL37 and HNP).

The highest bactericidal activity was found in the peptide ZP-2 (THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO), while its activity was detected not only in phosphate-buffered saline, but also in physiological saline against both gram-positive and gram-negative bacteria.

The presence of antifungal activity has not been established in any test sample.

Thus, the peptide of the invention ZP-1 (LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO), the peptide Zp-2 (THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO) to varying degrees , but to a degree sufficient for bactericidal activity, contribute to its increase when they are dissolved in physiological saline.

It is known that physiological saline is neutral in comparison with a buffer solution in relation to a living organism; therefore, the use of peptides ZP-1 (LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO) and ZP-2 (THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO), in solution with physiological saline, exhibiting bactericidal activity, does not exhibit possible negative effects of the buffer solution.

Example 1. A comparative assessment of bactericidal activity against St. Aureus 155 peptide ZP-1 at a concentration of 10 mg / ml in a buffer solution, compared with LL37 (prototype) at a concentration of 10 mg / ml in a buffer solution. The diameter of the zone of inhibition of growth of microorganisms was 10.0 and 19.0 mm, respectively.

Example 2. A comparative assessment of bactericidal activity against E. coli 2290 peptide ZP-2 at a concentration of 30 mg / ml in a buffer solution, compared with HNP (prototype) at a concentration of 30 mg / ml in a buffer solution. The diameter of the zone of inhibition of growth of microorganisms was 18.0 and 19.0 mm, respectively.

The inventive method increases the bactericidal activity against gram-positive and gram-negative bacteria in physiological saline, which is preserved in buffer media, in which synthetic peptides having the structure LYS GLY PRO LEY THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO and THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO, with a molecular weight of 1794 and 1397 daltons, respectively, physiological saline at the rate of 10–30 mg of synthetic peptides, 1 ml of physiological saline is used as a medicinal base.

Claims (1)

A method of increasing bactericidal activity, which consists in acting on gram-positive and gram-negative bacteria with peptides used in a drug basis, uses synthetic peptides having the structure LYS GLY PRO LEU THR NLE NLE ALA SER HIS TYR LYS GLN HIS CYS PRO and THR NLE NLE ALA as peptides SER HIS TYR LYS GLN HIS CYS PRO, with a molecular weight of 1794 and 1397 Yes, respectively, physiological saline at the rate of 10–30 mg of synthetic peptides, 1 ml of physiological saline is used as a medicinal base.