EP4132945A2 - Lipopeptides antibactériens, composition pharmaceutique et composition cosmétique les comprenant, et leurs utilisations - Google Patents

Lipopeptides antibactériens, composition pharmaceutique et composition cosmétique les comprenant, et leurs utilisations

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Publication number
EP4132945A2
EP4132945A2 EP21727925.6A EP21727925A EP4132945A2 EP 4132945 A2 EP4132945 A2 EP 4132945A2 EP 21727925 A EP21727925 A EP 21727925A EP 4132945 A2 EP4132945 A2 EP 4132945A2
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EP
European Patent Office
Prior art keywords
dab
leu
ddab
lipopeptide
dphe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21727925.6A
Other languages
German (de)
English (en)
Inventor
Slawomir SEK
Dagmara TYMECKA
Joanna JUHANIEWICZ-DEBINSKA
Dariusz BARTOSIK
Robert LASEK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Uniwersytet Warszawski
Original Assignee
Uniwersytet Warszawski
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Uniwersytet Warszawski filed Critical Uniwersytet Warszawski
Publication of EP4132945A2 publication Critical patent/EP4132945A2/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Antibacterial lipopeptides are, pharmaceutical composition and cosmetic composition comprising them, and uses thereof
  • the present invention relates to novel synthetic, linear and branched lipopeptides, pharmaceutical compositions comprising such synthetic, linear and/or branched lipopeptides, and cosmetic compositions comprising such synthetic, linear and/or branched lipopeptides, as well as such linear and branched lipopeptides for use as medicines, especially in treating bacterial infections.
  • Lipopeptides include daptomycin, which is a natural, branched, cyclic lipopeptide antibiotic of non-ribosomal origin. This lipopeptide shows a strong bactericidal activity in vitro and in vivo against Gram-positive bacteria that can cause serious and life-threatening diseases, as described, for example, in Tally et al. (Tally, F.P. et a/., "Daptomycin: a Novel Agent for Gram positive Infections”, Exp. Opin. Invest. Drugs 8 (1999) 1223-1238).
  • a group of lipopeptides that show significant potential as useful antibiotics includes the lipopeptides described, for example, in U.S. Patent No. 6,911,525 and U.S. Patent Application No. 2015/0080292 A1.
  • An object of the present invention is to provide synthetic lipopeptides that show antimicrobial activity against Grampositive and Gram-negative bacteria, including antibiotic-resistant bacteria, as well as pharmaceutical and cosmetic compositions comprising them.
  • X 1 is a residue selected from FA-DDab-Dab (SEQ ID NO.: 1) and H-Dab(FA-DDab) (SEQ ID NO.: 3), wherein when X 1 is FA-DDab-Dab then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, and 4-methylnonanoic acid, when X 1 is H-Dab(FA-DDab) then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, 4-methylnonanoic acid, and 4-methylhexanoic acid;
  • X 2 is a leucine, alanine, isoleucine or valine residue
  • X 3 is a D-phenylalanine, D-tyrosine, or D-tryptophan residue
  • X 4 is a leucine, alanine, isoleucine or valine residue.
  • X 1 is FA-DDab-Dab
  • X 2 is a leucine residue
  • X 3 is a D-phenylalanine residue
  • X 4 is a leucine residue.
  • Such a lipopeptide has a linear structure and can be represented by the following formula:
  • such a lipopeptide according to the invention is selected from the group consisting of the following lipopeptides:
  • X 1 is H-Dab(FA- DDab)
  • X 2 is a leucine residue
  • X 3 is a D-phenylalanine residue
  • X 4 is a leucine residue.
  • Such a lipopeptide has a branched structure and can be represented by the following formula:
  • such a lipopeptide according to the invention is selected from the group consisting of: H-Dab(CH 3 (CH 2 ) 9 CH 2 CO-DDab)-Dab-Leu-DPhe-Dab-Dab-Leu-NH 2 (condensed formula 4),
  • a further subject matter of the invention is a pharmaceutical composition, characterized in that it comprises a lipopeptide of the general formula:
  • X 1 is a residue selected from FA-DDab-Dab and H-Dab(FA-DDab), wherein when X 1 is FA-DDab-Dab then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, and 4-methylnonanoic acid, when X 1 is H-Dab(FA-DDab) then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, 4-methylnonanoic acid, and 4-methylhexanoic acid;
  • X is a leucine, alanine, isoleucine or valine residue; is a D-phenylalanine, D-tyrosine or D-tryptophan residue;
  • X 4 is a leucine, alanine, isoleucine or valine residue.
  • the pharmaceutical composition according to the invention comprises a lipopeptide of the above-indicated general formula according to the invention, wherein X 1 is FA-DDab-Dab, is a leucine residue, is a D- phenylalanine residue, and X 4 is a leucine residue.
  • the pharmaceutical composition according to the invention comprises a lipopeptide selected from the group consisting of:
  • the pharmaceutical composition according to the invention comprises a lipopeptide of the above-indicated general formula according to the invention, wherein X 1 is H-Dab(FA-DDab), is a leucine residue, is a D- phenylalanine residue, and X 4 is a leucine residue.
  • the pharmaceutical composition according to the invention comprises a lipopeptide selected from the group consisting of: H-Dab(CH 3 (CH 2 ) 9 CH 2 CO-DDab)-Dab-Leu-DPhe-Dab-Dab-Leu-NH 2 (condensed formula 4),
  • the pharmaceutical composition according to the invention preferably comprises 0.1 to 99% by weight of at least one of the above-described lipopeptides according to the invention.
  • the pharmaceutical composition according to the invention further comprises a pharmaceutically acceptable carrier and/or diluent and/or adjuvant and/or excipient.
  • the pharmaceutical composition according to the invention comprises corn starch and/or gelatin and/or lactose and/or sucrose and/or microcrystalline cellulose and/or kaolin and/or mannitol and/or dicalcium phosphate and/or sodium chloride and/or alginic acid.
  • the pharmaceutical composition according to the invention is in a form suitable for oral and/or intravenous and/or intramuscular and/or subcutaneous and/or parenteral administration.
  • the pharmaceutical composition according to the invention is in a form of a tablet and/or a capsule and/or an elixir and/or a suspension and/or a syrup and/or a gel and/or a cream and/or an ointment.
  • the pharmaceutical composition according to the invention further comprises at least one other antimicrobial agent.
  • a further subject matter of the invention is a cosmetic composition, characterized in that it comprises a lipopeptide of the general formula:
  • X 1 is selected from FA-DDab-Dab and H-Dab(FA-DDab), wherein when X 1 is FA-DDab-Dab then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, and 4-methylnonanoic acid, when X 1 is H-Dab(FA-DDab) then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, 4-methylnonanoic acid, and 4-methylhexanoic acid;
  • X is a leucine, alanine, isoleucine or valine residue; is a D-phenylalanine, D-tyrosine or D-tryptophan residue;
  • X 4 is a leucine, alanine, isoleucine or valine residue.
  • the cosmetic composition according to the invention comprises a lipopeptide of the above-indicated general formula according to the invention, wherein X 1 is FA-DDab-Dab, X 2 is a leucine residue, X 3 is a D- phenylalanine residue and X 4 is a leucine residue.
  • the cosmetic composition according to the invention comprises a lipopeptide selected from the group consisting of:
  • the cosmetic composition according to the invention comprises a lipopeptide of the above-indicated general formula, wherein X 1 is H-Dab(FA-DDab), X 2 is a leucine residue, X 3 is a D-phenylalanine residue and X 4 is a leucine residue.
  • the cosmetic composition according to the invention comprises a lipopeptide selected from the group consisting of:
  • the cosmetic composition according to the invention contains 0.1 to 99% by weight of at least one of the lipopeptides according to the invention.
  • the cosmetic composition according to the invention comprises a cosmetic carrier.
  • the cosmetic composition according to the invention is in a form of a gel or a cream or a suspension.
  • a further subject matter of the invention is any of the lipopeptides according to the invention, as defined above, for use as a medicament.
  • a further subject matter of the invention is any of the lipopeptides according to the invention, as defined above, for use in the treatment or prophylaxis of a bacterial infection.
  • any lipopeptide according to the invention for use in the treatment or prophylaxis of a bacterial infection is caused by a Gram-positive bacterium.
  • the bacterial infection is caused by a bacterium of the genus Enterococcus or Staphylococcus.
  • the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.
  • bacterial infection is caused by a Gram-negative bacterium.
  • the bacterial infection is caused by a bacterium of the genus Klebsiella or Pseudomonas.
  • the bacterial infection is caused by Klebsiella pneumoniae or Pseudomonas aeruginosa.
  • any lipopeptide according to the invention for use in the treatment or prophylaxis of a bacterial infection is caused by an antibiotic-resistant bacterium.
  • At least one lipopeptide is used selected from the group consisting of:
  • the present invention provides novel synthetic, linear and branched lipopeptides, pharmaceutical compositions comprising such synthetic, linear and/or branched lipopeptides, cosmetic compositions comprising such synthetic, linear and/or branched lipopeptides, as well as such linear and branched lipopeptides for use as medicines, especially in the treatment of bacterial infections.
  • the synthetic lipopeptide according to the invention is represented by the general formula:
  • X 1 is a residue selected from FA-DDab-Dab (SEQ ID NO.: 1) and H-Dab(FA-DDab) (SEQ ID NO.: 3), wherein when X 1 is FA-DDab-Dab then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, and 4-methylnonanoic acid, and the lipopeptide has a linear structure, when X 1 is H-Dab(FA-DDab) then FA is a fatty acid residue selected from the group comprising n-dodecanoic acid, n- decanoic acid, 4-methylnonanoic acid and 4-methylhexanoic acid, and the lipopeptide has a branched structure;
  • X is a leucine, alanine, isoleucine or valine residue - i.e. an aliphatic amino acid
  • X is a D-phenylalanine, D-tyrosine or D-tryptophan residue - i.e. an aromatic amino acid residue
  • X is a leucine, alanine, isoleucine or valine residue.
  • a lipopeptide is a molecule consisting of a lipid residue, i.e. a fatty acid (FA) residue linked to a peptide residue.
  • a lipid residue i.e. a fatty acid (FA) residue linked to a peptide residue.
  • FA fatty acid
  • a standard nomenclature in form of three-letter abbreviations known in the art is used, for example, a L-phenylalanine amino acid residue is designated as "Phe,” a D-phenylalanine residue as "DPhe,” L-leucine as "Leu,” L-2,4-diaminobutyric acid as “Dab,” and D-2,4- diaminobutyric acid as "DDab.”
  • the sequence listing shows the amino acid sequences of the peptide portion of the lipopeptides according to the invention.
  • Preferred linear lipopeptides according to the present invention can be represented by the following formula:
  • the lipopeptides according to the invention may be obtained by any synthetic method known in the prior art.
  • the mentioned synthesis method may include liquid-phase or solid-phase synthesis (see e.g. Sewald, N. and Jakubke, H.-D. (2003), Peptide Synthesis. In Peptides: Chemistry and Biology (eds. N. Sewald and H.-D. Jakubke) doi:10.1002/352760068X.ch4 and Peptide synthesis and applications. ed./Knud J. Jensen; Pernille T. Shelton; Soren L. Pedersen. Humana Press, 2013, (Methods in Molecular Biology, Vol. 1047).
  • An exemplary method of preparing the lipopeptides according to the invention is provided in the Examples section below.
  • the lipopeptides according to the invention both linear and branched, have an amphiphilic structure, and due to the presence of 2,4-diaminobutyric acid residues, under physiological pH (about 7.4) conditions, they have an excess positive charge, which facilitates their interaction with the bacterial cell membrane.
  • the pharmaceutical composition according to the invention comprises any linear or branched lipopeptide according to the invention preferably selected from the group of lipopeptides of condensed formulae 1-7.
  • the composition comprises 0.1 to 99% by weight of any of at least one lipopeptide according to the invention, preferably selected from the group comprising lipopeptides of condensed formulae 1-7.
  • the pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier and/or diluent and/or adjuvant and/or excipient, as known in the art.
  • the pharmaceutical composition may comprise corn starch and/or gelatin and/or lactose and/or sucrose and/or microcrystalline cellulose and/or kaolin and/or mannitol and/or dicalcium phosphate and/or sodium chloride and/or alginic acid.
  • an active agent such as an antimicrobial agent
  • at least one pharmaceutically acceptable carrier and/or diluent and/or adjuvant and/or excipient are known in the art.
  • the pharmaceutical composition according to the invention further comprises at least one other antimicrobial agent, such as an antibiotic.
  • antimicrobial agents that can be a component of the pharmaceutical composition according to the invention may be selected, i.a., from: b-lactam antibiotics such as, for example, penicillins, cephalosporins; peptide antibiotics such as, for example, polymyxins (e.g.
  • colistin gramicidin
  • bacitracin fusafungin
  • glycopeptide antibiotics such as, for example, vancomycin, oritavancin
  • aminoglycoside antibiotics such as, for example, streptomycin, gentamicin, amikacin
  • tetracycline antibiotics such as, for example, doxycycline
  • macrolide antibiotics such as, for example, erythromycin, clarithromycin
  • lincosamide antibiotics such as, for example, clindamycin, lincomycin
  • amphenicol antibiotics such as, for example, chloramphenicol
  • rifamycin antibiotics such as, for example, rifampicin and rifaximin.
  • the pharmaceutical composition according to the invention is in a form suitable for oral and/or intravenous and/or intramuscular and/or subcutaneous and/or parenteral administration, preferably in the form of a tablet and/or a capsule and/or an elixir and/or a suspension and/or a syrup and/or or a gel and/or a cream and/or an ointment.
  • Methods for preparing such forms of pharmaceutical compositions are known in the art of the present invention.
  • the cosmetic composition according to the invention comprises any linear or branched lipopeptide according to the invention, preferably selected from the group comprising lipopeptides of condensed formulae 1-7.
  • the cosmetic composition according to the invention may have any cosmetically acceptable dosage form.
  • the cosmetic composition may comprise 0.1 to 99% by weight of any of at least one lipopeptide according to the invention, preferably selected from the group of condensed formulae 1-7.
  • the cosmetic composition according to the invention optionally comprises a cosmetic carrier.
  • Cosmetic carriers are known in the art.
  • semi-solid or liquid polyols can be used, as well as fats, e.g. in form of micelles or liposomes.
  • the cosmetic composition is in the form of a gel, cream or suspension. Methods for preparing cosmetic compositions are known in the art.
  • the invention also includes synthetic, linear and branched lipopeptides according to the invention, preferably of condensed formulae 1-7, for use as a medicament.
  • the invention also includes synthetic, linear and branched lipopeptides according to the invention, preferably of condensed formulae 1-7, for use in the treatment of bacterial infections, preferably caused by Gram-positive bacteria, more preferably of the genus Staphylococcus.
  • the advantage of the synthetic linear and branched lipopeptides according to the invention is their mechanism of action, which is based on direct interaction with the bacterial cell membrane, which in turn reduces the risk of drug resistance. Due to a different mechanism of action to antibiotics known in the art, the lipopeptides according to the invention will show effectiveness against bacteria resistant to antibiotics with a different mechanism of action than the lipopeptides according to the invention. Additionally, the synthetic, linear and branched lipopeptides according to the invention are simple and easy to synthesize structures, what significantly reduces their preparation cost.
  • the synthetic, linear and branched lipopeptides according to the invention are also characterized by an appropriate solubility in an aqueous medium, which significantly extends the range of possibilities regarding the dosage form of the pharmaceutical or cosmetic composition according to the invention.
  • the synthetic, linear and branched lipopeptides according to the invention are also characterized by an increased metabolic stability, which ensures an additional benefit in the form of a significant extension of their activity time, and thus additionally increases their effectiveness.
  • Figure 1 shows an exemplary scheme for the preparation of a synthetic linear lipopeptide according to the invention (condensed formula 2).
  • Figure 2 shows an exemplary scheme for the preparation of a synthetic branched lipopeptide according to the invention (condensed formula 5).
  • Figure 3 shows the effect of the lipopeptide of condensed formula 1 according to the invention on the growth dynamics of Pseudomonas aeruginosa (panel A) and Staphylococcus aureus (panel B) strains, shown as relative culture optical density as a function of time.
  • Figure 4 shows the effect of the lipopeptide of condensed formula 2 according to the invention on the growth dynamics of Pseudomonas aeruginosa (panel A) and Staphylococcus aureus (panel B) strains, shown as relative culture optical density as a function of time.
  • Figure 5 shows the effect of the lipopeptide of condensed formula 3 according to the invention on the growth dynamics of Pseudomonas aeruginosa (panel A) and Staphylococcus aureus (panel B) strains, shown as relative culture optical density as a function of time.
  • Figure 6 shows the effect of a reference lipopeptide on the growth dynamics of Pseudomonas aeruginosa (panel A) and Staphylococcus aureus (panel B) strains, shown as relative culture optical density as a function of time.
  • the lipopeptides both linear and branched, were obtained by solid-phase peptide synthesis according to the Fmoc/tBu synthesis strategy on a polymer support, which was the Fmoc-Rink Amide AM amide resin (with 0.55 mmol/g loading, Activotec, UK).
  • the synthesis was started by weighing out 365 mg of the resin and placing it in a syringe reactor, then adding 5 mL of N,N-dimethylformamide (DMF, Sigma-Aldrich) and shaking for 2 hours to swell the resin. Then the following steps were carried out:
  • the Kaiser test was used to evaluate the effectiveness of the Fmoc-deprotection step. Utilizing the standard procedure for performing this test, i.e. suspending a sample of the resin particles in a mixture of A-C solutions (3-5 drops each) and heating to approx. 100°C, using starting solutions composed of: 5 g of ninhydrin in 100 mL of ethanol (solution A); 80 g of phenol in 20 mL of ethanol (solution B); 2 mL of 0.001 M aqueous KCN solution in 98 mL of pyridine (solution C). The dark blue color of both particles and solution indicated the presence of a free amino group.
  • the next step was to perform a condensation reaction between the amino acid derivative (Fmoc-Xaa)-OH and the amino group of the resin.
  • the appropriate amounts of reagents were weighed and metered out into the vessel (molar ratio in relation to the resin loading):
  • the Kaiser test was performed (according to 2), this time to evaluate the progress/completion of the condensation reaction.
  • the negative test result i.e. colorless both particles and solution, indicated the complete attachment of Fmoc-protected amino acid to the resin, i.e. the condensation reaction was complete.
  • the amino acid derivative coupling step was repeated using a double excess of reagents (Fmoc-Xaa- OH/DIC/Oxyma molar ratio was 1:1:1).
  • the penultimate step of the synthesis was the attachment of fatty acid (R-COOH, Merck).
  • R-COOH fatty acid
  • Merck fatty acid
  • washed peptidyl-resins were dried in a vacuum desiccator for 30 minutes.
  • the final step of the syntheses was the step of acidolytic cleavage of the peptide from the polymer support, in which Reagent B was used: TFA/HiO/phenol/TIPS (88:5:5:2, v/v), (where: TFA - trifluoroacetic acid, TIPS - triisopropylsilane).
  • Reagent B was added to the dried peptidyl-resin in a ratio of approx. 10 mL per 1 g of peptidyl- resin. The mixture was shaken for 2-4 hours. After this, the resin was filtered off, washed with an additional small amount of TFA, and the resulting filtrate was concentrated by evaporator. Cold diethyl ether (chilled in dry ice) was poured over the residue and it was allowed to stand for 24 hours in the refrigerator to precipitate the peptide. Next, after 24 hours, the crude peptide was isolated by centrifugation.
  • the analytical HPLC was performed on Prominence modular liquid chromatograph by Shimadzu with a DAD detector. A Luna 5 ⁇ m, C18 (2) 100A reverse-phase column, 250 x 4.6 mm, was used. Semi-preparative RP-HPLC were performed on a Prominence LC-20AP modular liquid chromatograph with a UV-VIS detector. A Luna 5 ⁇ m, C18 column, 150 x 10 mm, was used. Identification of the obtained lipopeptides was performed on the basis of mass spectra obtained on the Shimadzu LCMS-IT-TOF instrument, which was equipped with an electrospray ion source (ESI) with an ion trap (IT) and a time-of-flight (TOF) analyzer. Prior to the purification step of a given peptide, RP- HPLC analysis of the crude product was performed in order to optimize the separation conditions for the given crude peptide mixture.
  • ESI electrospray ion source
  • IT ion trap
  • TOF time-of
  • 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, and Enterococcus faecalis ATCC 14506.
  • Gram-negative bacterial strains Escherichia coli ST2- 8624 0157:H7, Pseudomonas aeruginosa PA01 PCM 499, Klebsiella pneumoniae PCM 1, Salmonella sv. Typhimurium TT622, and Yersinia enterocolitica PCM 2081. All strains were grown in LB (lysogeny broth) medium in a conventional manner.
  • LB medium Ten mL of LB medium was inoculated with material obtained from a single colony and the culture was allowed to grow overnight at 30°C with shaking. Next, the optical density of the overnight cultures (at 600 nm) was then adjusted to 0.05 by dilution in a fresh portion of LB medium. The test compounds were dissolved in water. Serial dilutions of compounds were prepared in LB medium ranging from 5 to 50 ⁇ g/mL (final concentrations). The experiment was performed by adding 100 ⁇ L of each of the prepared dilutions to 100 ⁇ L of the diluted overnight bacterial culture in the wells of a 96-well microtiter plate.
  • the MIC was defined as the lowest concentration of test compound needed to inhibit bacterial growth evaluated after 24 h incubation at 30°C with shaking (final optical density at 600 nm not greater than 0.05). Optical density measurements were performed using TECAN Sunrise plate reader. Data were obtained from three independent experiments. The obtained results are summarized in Table 3.
  • the results of the measurements summarized in Table 3 indicate that all the tested lipopeptides show varying activity against the tested bacterial strains.
  • the lipopeptides according to the invention show activity against both Grampositive and Gram-negative bacterial strains.
  • the reference lipopeptide not being the subject matter of the invention, and placed in the table just for comparative purposes, shows little activity against only two of the eight strains analyzed.
  • the obtained results indicate a certain degree of selectivity of the lipopeptides according to the invention for Gram-positive strains of Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, for which definitely the highest activity of the tested lipopeptides was observed, i.e. the lowest values of the minimum inhibitory concentration (MIC).
  • the activity of the tested lipopeptides against Yersinia enterocolitica PCM 2081 and Salmonella sv. Typhimurium TT622 strains is scarce.
  • the starting solutions of each of the tested lipopeptides were prepared at a concentration of 70 pmol/mL by dissolving previously weighed out portions of compounds in the appropriate volume of the internal standard solution.
  • the measuring systems were mixed at 37°C and 450 rpm using a Eppendorf Thermomixer R.
  • the protocol for measuring lipopeptide degradation in human blood serum included:
  • All tested lipopeptides according to the invention undergo enzymatic degradation in human blood serum, nevertheless they are characterized by an increased metabolic stability, and their half-lives are above 27 hours, which is the time observed for the reference lipopeptide.
  • the shortest half-lives have the lipopeptides comprising the shortest fatty acid chains.
  • all lipopeptides according to the invention are characterized by the longer half-lives than the reference lipopeptide and therefore have an increased metabolic stability.

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Abstract

La présente invention concerne des lipopeptides antimicrobiens synthétiques ayant une structure linéaire et des lipopeptides antimicrobiens ayant une structure ramifiée, des compositions pharmaceutiques comprenant de tels lipopeptides, des compositions cosmétiques comprenant de tels lipopeptides et des lipopeptides destinés à être utilisés en tant que médicament, ainsi que des lipopeptides destinés à être utilisés dans le traitement ou la prophylaxie d'une infection bactérienne.
EP21727925.6A 2020-04-08 2021-04-08 Lipopeptides antibactériens, composition pharmaceutique et composition cosmétique les comprenant, et leurs utilisations Pending EP4132945A2 (fr)

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PL433503A PL433503A1 (pl) 2020-04-08 2020-04-08 Lipopeptydy o działaniu przeciwbakteryjnym, kompozycja farmaceutyczna i kompozycja kosmetyczna je zawierająca oraz zastosowania
PCT/IB2021/052911 WO2021205372A2 (fr) 2020-04-08 2021-04-08 Lipopeptides antibactériens, composition pharmaceutique et composition cosmétique les comprenant, et leurs utilisations

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EP4132945A2 true EP4132945A2 (fr) 2023-02-15

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WO2021205372A3 (fr) 2022-01-20
WO2021205372A2 (fr) 2021-10-14
PL433503A1 (pl) 2021-10-11

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