Lipid oligomeric urea, pharmaceutical composition and lipid oligomeric urea for use as medicament

Abstract

The subject of the present invention is a lipooligomeric urea of formula (1): FA-Pheu-Xaa1u-Xaa2u-Leuu-Xaa3u-NH2 (1) wherein FA is a linear or branched carboxylic acid residue containing from 6 to 15 carbon atoms in the alkyl chain; x < aa1 > u is Lysu, or Dabu, or Argu; xaa2u is Lysu, or Dabu, or Leuu, or Argu, or Xaa2u is Ysu, or Dabu, or Leuu, or Argu; in the formula, Xaa3u is Lysu, or Dabu, or Leuu, or Argu. The invention also relates to a pharmaceutical composition containing the lipooligomeric urea of formula (1).

Description

[0001] The present invention relates to lipooligourea, a pharmaceutical composition containing lipooligourea and use of lipooligourea as a drug for treating bacterial infection.

[0002] The increasing incidence of bacterial resistance has become a major clinical problem, resulting in a growing interest in compounds that offer effective alternatives while exhibiting a different mechanism of action than traditional antibiotics. A novel group of compounds that has the potential to meet these requirements are the lipid oligoureas described below.

[0003] In most cases, commercially available traditional antibiotics show specific effects based on the biochemical processes occurring in the destruction of bacterial cells. For example, they affect the activity of enzymes involved in cell wall synthesis, destroy metabolic pathways or damage DNA. Antibiotics based on peptides and lipopeptides are also known antibiotics; their action specificity is low, and based on the mechanism of using such compounds to penetrate and irreversibly destroy the ability of bacterial cell membrane structure (for example, see Alborn et al., Antimicrob.Agents Chemother.1991,35,2282, and Kang et al., Journal ofMicrobiology vol.2017,55,1). The membrane-dissolving properties of antibiotic peptides and lipopeptides significantly reduce the possibility of bacteria developing resistance to such compounds, which is due to non-specific mechanisms of action. There are currently some drugs on the market, whose active antibiotic ingredients are, for example, lipopeptides. Such drugs can include polymyxin or daptomycin. Daptomycin is a natural branched cyclic antibiotic lipopeptide of non-ribosomal origin. This lipopeptide exhibits strong bactericidal activity against Gram-positive bacteria in vitro and in vivo, which can cause serious life-threatening diseases, such as described in the following literature: Tally, FP et al., "Daptomycin: a Novel Agent for Gram positive Infections," Exp. Opin. Invest. Drugs 8: 1223-1238 (1999). Most of the lipopeptides described in the literature (such as daptomycin) are of natural origin, derived from bacteria or other organisms, which requires the cost of separation from biological materials. In addition, most of the lipopeptides currently used in medicine (such as daptomycin or polymyxin) have a cyclic, relatively complex structure, which in turn complicates potential synthesis and further modification. However, there are also examples of synthetic compounds belonging to the class of lipopeptides known in the literature which show great potential as useful antibiotics and which are at the same time based on non-cyclic (ie linear) structures (see for example US 6,911,525; US 2015 / 0080292 A1; WO 2021 / 064593 A1).

[0004] The purpose of the present invention is to provide a new class of compounds - lipid oligoureas, which have a non-cyclic structure and show antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.

[0005] This goal was achieved by developing a new class of lipid oligoureas with antimicrobial properties.

[0006] Therefore, the subject of the present invention is a lipid oligourea of ​​the general formula 1

[0007] FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1)

[0008] in

[0009] FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain;

[0010] Xaa1 u It's Lys u , or Dab u , or Arg u ;

[0011] XAa2 u It's Lys u , or Dab u , or Leu u , or Arg u ;

[0012] XAa3 u It's Lys u , or Dab u , or Leu u , or Arg u .

[0013] Preferably, the lipid oligourea is selected from the group consisting of:

[0014] LOU1

[0015]

[0016] LOU2

[0017]

[0018] LOU3

[0019]

[0020] LOU4

[0021]

[0022] A subject of the present invention is also a lipid oligourea as defined above for use as a medicament.

[0023] Preferably, it is used to treat or prevent bacterial infections.

[0024] Preferably, the bacterial infection is caused by Gram-positive or Gram-negative bacteria.

[0025] Preferably, the bacterial infection is caused by Staphylococcus bacteria.

[0026] Preferably, the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

[0027] Preferably, the bacterial infection is caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA).

[0028] The subject of the present invention is a lipolyurea of ​​formula LOU2 or LOU3 for use in the treatment or prevention of bacterial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains.

[0029] Another subject of the present invention is a pharmaceutical composition containing a lipid oligourea of ​​the general formula 1

[0030] FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1)

[0031] in

[0032] FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain;

[0033] Xaa1 u It's Lys u , or Dab u , or Arg u ;

[0034] XAa2 u It's Lys u , or Dab u , or Leu u , or Arg u ;

[0035] XAa3 u It's Lys u , or Dab u , or Leu u , or Argu .

[0036] Preferably, the pharmaceutical composition contains a lipid oligourea selected from the group consisting of:

[0037] LOU1:

[0038]

[0039] LOU2:

[0040]

[0041] LOU3:

[0042]

[0043] LOU4:

[0044]

[0045] Preferably, the pharmaceutical composition contains 0.1% to 99% by weight of at least one lipid oligourea.

[0046] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier and / or diluent and / or adjuvant and / or excipient.

[0047] Preferably, the pharmaceutical composition contains corn starch and / or gelatin and / or lactose and / or sucrose and / or microcrystalline cellulose and / or kaolin and / or mannitol and / or calcium hydrogen phosphate and / or sodium chloride and / or alginic acid.

[0048] Preferably, the pharmaceutical composition is in a form suitable for oral administration and / or intravenous administration and / or intramuscular administration and / or subcutaneous administration and / or parenteral administration and / or rectal administration and / or topical administration.

[0049] Preferably, the pharmaceutical composition is in the form of tablets and / or capsules and / or elixirs and / or suspensions and / or syrups and / or gels and / or creams and / or ointments and / or suppositories and / or aerosols and / or drops.

[0050] Preferably, the pharmaceutical composition further comprises at least one other antibacterial agent.

[0051] Unlike most traditional antibiotics (which have specific effects as mentioned above), lipid oligoureas show the ability to bind to and enter bacterial cell membrane structures and cause irreversible damage to them. Since the specificity of their activity is lower than that of traditional drugs, the possibility of developing resistance to this type of compound is less. Compared with antibiotic peptides and lipopeptides, the important characteristics and advantages of lipid oligoureas are also the low sensitivity of oligourea chains to proteolytic degradation. This process can significantly reduce the activity of peptides and lipopeptides, while in the case of synthetic peptidomimetics (such as lipid oligoureas), this process will be significantly eliminated. Therefore, the scheme proposed in this article involves a new class of antibacterial compounds, namely lipid oligoureas. The lipid oligourea is composed of an oligourea portion comprising five urea residues connected to a straight or branched carboxylic acid residue. For the purpose of this article, the standard nomenclature of the three-letter amino acid abbreviation form (wherein the "u" subscript indicates the urea nature of the residue) is used to refer to urea residues, which are analogs of single amino acids. The structure of the lipid oligourea can be represented by the general formula:

[0052] FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1)

[0053] wherein FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain;

[0054] Xaa1 u It's Lys u , or Dab u , or Arg u ;

[0055] XAa2 u It's Lys u , or Dab u , or Leu u , or Arg u ;

[0056] XAa3 u It's Lys u , or Dab u , or Leu u , or Arg u .

[0057] The developed lipid oligoureas have an amphiphilic structure, which allows them to be incorporated into the lipid bilayer that constitutes the framework of the cell membrane and to bind to the lipid bilayer due to the presence of positively charged urea residues, such as Lys u , Dab u or Arg u, which have an excess of positive charges under physiological pH conditions, which further facilitates their interaction with bacterial cell membranes, which usually have an excess of negative charges due to their specific lipid composition (i.e. the presence of lipids from phosphatidylglycerol groups).

[0058] The pharmaceutical composition according to the present invention contains any lipid oligourea selected from the group of lipid oligoureas of general formula (1). Preferably, the composition contains 0.1% by weight to 99% by weight of lipid oligourea selected from the group of lipid oligoureas comprising general formula (1). The pharmaceutical composition may contain a pharmaceutically acceptable carrier and / or a diluent and / or an adjuvant and / or an excipient. For example, but not limited to the above substances, the pharmaceutical composition may contain corn starch and / or gelatin and / or lactose and / or sucrose and / or microcrystalline cellulose and / or kaolin and / or mannitol and / or calcium hydrogen phosphate and / or sodium chloride and / or alginic acid. Preferably, the pharmaceutical composition is in a form suitable for oral administration and / or intravenous administration and / or intramuscular administration and / or subcutaneous administration and / or parenteral administration and / or rectal administration and / or topical administration (e.g. on the skin, in the nose, in the ear), for example in the form of tablets and / or capsules and / or elixirs and / or suspensions and / or syrups and / or gels and / or creams and / or ointments and / or suppositories and / or aerosols and / or drops.

[0059] The present invention also comprises a lipooligourea of ​​general formula (1) for use as a medicament. Preferably, the lipooligourea of ​​general formula (1) is intended to treat bacterial infections, preferably caused by Gram-positive bacteria, in particular bacteria of the genus Staphylococcus.

[0060] The lipid oligourea according to the present invention can be obtained by any synthesis method known in the art. For example, the synthesis method may include liquid phase synthesis or solid phase synthesis.

[0061] The lipid oligourea according to the present invention has an amphiphilic structure and due to the presence of Lys u , Dab u or Arg u The urea residues of β-lactamases have excess positive charges under physiological pH conditions (about 7.4), which facilitates their interaction with bacterial cell membranes.

[0062] According to the present invention, lipid oligoureas can be used to treat bacterial infections due to their growth inhibition activity against Gram-positive bacteria (e.g., Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis) strains and Gram-negative bacteria (e.g., Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia enterocolitica). According to the present invention, lipid oligoureas show enhanced activity against Staphylococcus bacteria. Importantly, lipid oligoureas according to the present invention show significant growth inhibition activity against methicillin-resistant Staphylococcus aureus (MRSA) bacterial strains.

[0063] The advantage of the lipid oligourea according to the present invention is that it is based on a mechanism of action of direct interaction with the bacterial cell membrane, which in turn reduces the risk of drug resistance. In addition, the structure of the lipid oligourea according to the present invention is relatively simple, which significantly facilitates their synthesis. It can also ensure that they are fully dissolved in aqueous media, which significantly broadens the range of possibilities for the application form of the potential pharmaceutical composition.

[0064] To illustrate the present invention, examples are attached below, however, the examples should not be construed as limiting the scope of the present invention in any way. Example

[0065] Bacterial strains and culture media

[0066] All bacterial strains were obtained from the Polish Collection of Microorganisms (PCM) or the American Type Culture Collection (ATCC). Gram-positive bacterial strains: Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 14506. Methicillin-resistant Staphylococcus aureus (MRSA) bacterial strains: Staphylococcus aureus 401 / 5, Staphylococcus aureus 4811 / 13, Staphylococcus aureus 5288 / 13. Gram-negative bacterial strains: Escherichia coli ST2-8624O157:H7, Pseudomonas aeruginosa PAO1 PCM 499, Klebsiella pneumoniae PCM 1, Salmonella typhimurium TT622, and Yersinia enterocolitica PCM 2081. All strains were cultured in LB medium (lysogeny broth).

[0067] Synthesis of the lipid oligoureas tested according to the invention

[0068] Oligourea is obtained from succinimidyl carbamate derivatives substituted by 2-azidoethylamine at the 2-position. These building blocks are obtained from amino acids according to methods known in the literature (C. Douat-Casassus, K. Pulka, P. Claudon and G. Guichard, Org. Lett., 2012, 14, 3130-3133). Oligourea is solid phase prepared on NovaPEG type Rink amide resin with a loading of 0.45 mmol / g. 300 mg of resin is used for synthesis each time. Synthesis is carried out under microwave irradiation, and each step is verified by tetrachlorobenzoquinone test. For the coupling step of a single building block, 1.5 equivalents of the corresponding building block, 3 equivalents of DIPEA and 4 mL of DMF are used. The reaction is carried out in a microwave reactor under the following conditions: 50 ° C and 50 W for 30 minutes. Repeat the steps (1 or 2 times) until resin particles of suitable color are obtained in the tetrachlorobenzoquinone test. The step of reducing the azide group to an amine group was carried out with a THF solution of 1M trimethylphosphine (PMe3). 10 equivalents of PMe3 were used and reacted in a 7:3 1,4-dioxane:H2O solution. The reaction was carried out in a microwave reactor under the same conditions as the coupling step. The reduction reaction was repeated until resin particles of appropriate color were obtained in the tetrachlorobenzoquinone test. After the synthesis of the oligomeric part, a carboxylic acid (FA) residue was attached. 3.5 equivalents of acid, 3.5 equivalents of HBTU and 9 equivalents of DIPEA were used. The reaction was carried out in DMF at room temperature for 24 hours. The final compound was cleaved from the resin using a separation mixture consisting of TFA:TIS:H2O in a ratio of 95:2.5:2.5. The lipid oligourea was purified by semi-preparative HPLC using acetonitrile and water and 0.1% TFA as mobile phases. A gradient was selected for each compound to obtain the best possible separation (partitioning) and yield. The oligourea after HPLC purification was obtained in the form of a salt (ie, trifluoroacetate). The chloride ions were then exchanged with trifluoroacetate ions using DOWEX 1X8-100 type ion exchange resin, and the ion exchanged solution was lyophilized to obtain the final compound.

[0069]

[0070] The following section of this example describes the biological activity of an exemplary lipid oligourea having the formula: LOU1:

[0071]

[0072] LOU2:

[0073]

[0074] LOU3:

[0075]

[0076] LOU4:

[0077]

[0078] Determination of Minimum Inhibitory Concentration (MIC)

[0079] 10mL LB culture medium was inoculated with material from a single bacterial colony, and cultured overnight at 30°C with shaking. Then the optical density (at 600nm) of the overnight culture was reached to 0.05 by diluting the LB culture medium with a fresh portion. The test compound was dissolved in water. In the LB culture medium of 5μg / mL to 50μg / mL (final concentration) range, the compound was serially diluted. Experiments were conducted by adding 100μL of each prepared dilution to 100μL diluted overnight bacterial culture in the wells of 96-well titration plates. MIC was defined as the minimum concentration (the final optical density at 600nm was not more than 0.05) of the test compound required for inhibiting bacterial growth evaluated after 24 hours of shaking incubation at 30°C. Optical density measurement was performed using a TECAN Sunrise plate reader. Data were obtained from three independent experiments. Table 1 summarizes the results obtained.

[0080] Table 1

[0081]

[0082] MIC = minimum inhibitory concentration

[0083] nd = not determined, i.e. MIC value higher than 50 μg / mL

[0084] The measurement results summarized in Table 1 show that all tested lipid oligoureas show different activities to the tested bacterial strains. According to the lipid oligourea of ​​the present invention, both Gram-positive bacterial strains and Gram-negative bacterial strains show activity. The obtained results show that the lipid oligourea according to the present invention has a certain degree of selectivity to the Gram-positive bacterial strains of Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228, and for the above-mentioned bacterial strains, the activity of the lipid oligourea tested is the highest, i.e., the MIC value is the lowest. This trend is also reflected in the activity to methicillin-resistant Staphylococcus aureus (MRSA) strains. In this case, LOU2 and LOU3 show particularly high activity.

[0085] Exemplary pharmaceutical compositions according to the present invention

[0086] The composition of antibacterial ointment includes:

[0087] -40.0 g 1% boric acid aqueous solution;

[0088] - 29.9 g lanolin;

[0089] - 29.9 g Eucerin;

[0090] - 0.2 g of the lipid oligourea according to the invention.

[0091] Susceptibility of Lipo-oligoureas to Proteolytic Degradation

[0092] The stability of LOU1-LOU4 compounds in serum was studied using human serum without coagulation factors (obtained from Innovative Research, Inc., USA). The stock solution of lipid oligourea (0.168M) was diluted (1:69) in human serum to an incubation concentration of 2.4 μmol / ml for each lipid oligourea. The samples were then incubated at 37°C and 50 μL aliquots were taken at specified time intervals (0 minutes, 24 hours, 48 ​​hours, 72 hours, 96 hours).

[0093] In order to precipitate serum proteins, 200 μL ACN / H2O / FA ​​(89:10:1, v / v) were added to the collected samples to obtain a turbid mixture. These mixtures were vortexed at 3000 rpm for 1 minute, cooled in a refrigerator for 20 minutes, and then centrifuged at 4 ° C (14,000 rpm) for 15 minutes to precipitate protein. 150 μL supernatant was collected from each sample and 300 μL water was added before lyophilization. The lyophilized samples were then redissolved in 150 μL ACN / H2O / FA ​​(10:90:0.01, v / v) and analyzed using RP-HPLC, wherein a gradient of 10% solvent B in A was used for 5 minutes, and then a gradient of 10% to 97% solvent B in A was used for 30 minutes, wherein solvent A was a 0.1% TFA H2O solution, and solvent B was a 0.1% TFA CH3CN solution. H-Trp-OH and Z-Lys-OH internal standards were used in the analysis. Two independent experiments were performed for each sample and each experiment was performed once.

[0094] The sensitivity of LOU1-LOU4 compounds to proteolytic degradation was evaluated by incubating the lipid oligoureas in human serum at 37°C for a total of 96 hours. The progress of enzymatic degradation of each sample was monitored by RP-HPLC at 24-hour intervals, and the changes in the lipid oligourea peak area on the chromatogram were determined. The initial area at time t0 was recorded as 100%. After 96 hours, the largest decrease in peak area was observed for LOU1 (about 15%). However, this decrease may be due to the co-precipitation of lipid oligoureas with serum proteins, as we did not detect any new peaks corresponding to proteolytic degradation products. In contrast, the peak areas of the other compounds showed no significant changes (up to 8%), confirming the expected high resistance of lipid oligoureas to proteolytic degradation in serum. Figure 1 The time-dependent changes in the concentration of lipid oligourea are shown.

[0095] Figure 1 Shown is the time-dependent change in the amount of remaining lipoolureas that have been proteolytically degraded in human serum.

Claims

1. Lipid oligourea having general formula 1 FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1) FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain; Xaa1 u It's Lys u , or Dab u , or Arg u ; XAa2 u It's Lys u , or Dab u , or Leu u , or Arg u ; XAa3 u It's Lys u , or Dab u , or Leu u , or Arg u .

2. The lipid oligourea according to claim 1, selected from the group consisting of: LOU1 LOU2 LOU3 LOU4 3. The lipid oligourea as defined in claim 1 or 2 for use as a medicament.

4. The lipooligourea as defined in claim 1 or 2, for use in treating or preventing bacterial infection.

5. The lipid oligourea used according to claim 4, characterized in that The bacterial infection is caused by Gram-positive bacteria or Gram-negative bacteria.

6. The lipid oligourea used according to claim 4 or 5, characterized in that The bacterial infection is caused by Staphylococcus bacteria.

7. The lipid oligourea used according to claim 4, 5 or 6, characterized in that The bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

8. The lipid oligourea used according to claim 4 or 5, characterized in that The bacterial infection was caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA).

9. A lipoolurea of ​​formula LOU2 or LOU3 for use in the treatment or prevention of bacterial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains.

10. A pharmaceutical composition, characterized in that It contains a lipid oligourea of ​​the general formula 1 FA-Phe u -No1 u -No2 u -Leo u -No3 u -NH2(1) in FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain; Xaa1 u It's Lys u , or Dab u , or Arg u ; XAa2 u It's Lys u , or Dab u , or Leu u , or Arg u ; XAa3 u It's Lys u , or Dab u , or Leu u , or Arg u .

11. The pharmaceutical composition according to claim 10, characterized in that It contains a lipid oligourea selected from the group consisting of: LOU1 LOU2 LOU3 LOU4 12. The pharmaceutical composition according to claim 10 or 11, characterized in that It contains 0.1 to 99% by weight of at least one lipid oligourea.

13. The pharmaceutical composition according to any one of claims 10 to 12, characterized in that It contains pharmaceutically acceptable carriers and / or diluents and / or adjuvants and / or excipients.

14. The pharmaceutical composition according to claim 13, characterized in that It contains corn starch and / or gelatin and / or lactose and / or sucrose and / or microcrystalline cellulose and / or kaolin and / or mannitol and / or calcium hydrogen phosphate and / or sodium chloride and / or alginic acid.

15. The pharmaceutical composition according to any one of claims 10 to 14, characterized in that It is in a form suitable for oral administration and / or intravenous administration and / or intramuscular administration and / or subcutaneous administration and / or parenteral administration and / or rectal administration and / or topical administration.

16. The pharmaceutical composition according to any one of claims 10 to 15, characterized in that It is in the form of a tablet and / or capsule and / or elixir and / or suspension and / or syrup and / or gel and / or cream and / or ointment and / or suppository and / or aerosol and / or drops.

17. The pharmaceutical composition according to any one of claims 10 to 16, characterized in that It also contains at least one other antimicrobial agent.

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