WO2010113042A1 - Peptides cycliques antimicrobiens - Google Patents

Peptides cycliques antimicrobiens Download PDF

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Publication number
WO2010113042A1
WO2010113042A1 PCT/IB2010/001250 IB2010001250W WO2010113042A1 WO 2010113042 A1 WO2010113042 A1 WO 2010113042A1 IB 2010001250 W IB2010001250 W IB 2010001250W WO 2010113042 A1 WO2010113042 A1 WO 2010113042A1
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WIPO (PCT)
Prior art keywords
replaced
orn
leu
val
pro
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PCT/IB2010/001250
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English (en)
Inventor
Mark Overhand
Vanita Varsha Kapoerchan
Gijsbert Arie Van Der Marel
Herman Steven Overkleeft
Albert Johannes De Neeling
Daniel Noort
Annemiek Dorien Knijnenburg
Original Assignee
Universiteit Leiden
Nederlandse Organisatie Voor Toegepast- Natuurwetenschappelijk Onderzoek Tno
Rijksinstituut Voor Volksgezondheid En Milieu
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Publication of WO2010113042A1 publication Critical patent/WO2010113042A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/66—Gramicidins S, C; Tyrocidins A, B, C; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to antimicrobial cyclic peptides, which display a reduced hemolytic activity and optionally antimicrobial activity in comparison to parent antimicrobial cyclic peptides from which they are derived.
  • the parent cyclic peptide is gramicidin S.
  • a method of providing novel derivative antimicrobial cyclic peptides which have been developed from a parent antimicrobial cyclic peptide and which display a reduced haemolytic activity and/or antimicrobial activity compared to the parent molecule from which they are derived.
  • the cyclic decapeptide gramicidin S (GS, c(Pro-Val-Orn-Leu-DPhe)2, produced by Bacillus brevis belongs to the class of cationic antimicrobial peptides.
  • the peptide is able to disrupt the bacterial cell membrane with bacterial cell lysis and death as a result.
  • GS displays considerable activity against both Gram-positive and Gram-negative bacteria.
  • the ability of GS to disrupt the integrity of lipid bi-layer(s) is attributed to both the basic and amphiphilic nature of its rigid cyclic ⁇ -hairpin molecular structure.
  • the fact that the enantiomer of GS is equally active underscores the current thinking that the cell membrane, and not a specific gene product, is targeted by GS.
  • GS The structure of GS is amphiphilic with the side chains of the two Om residues on one side of the molecule and the hydrophobic side chains of the two VaI and Leu residues residing on the opposing face.
  • Drawback of GS is its toxicity towards human cells, limiting its antibiotic use to topical applications. This toxicity is associated with the non- discriminative property of GS to disrupt both bacterial- and mammalian cell membranes.
  • GS was the subject of extensive structure-activity studies, in which amino acids throughout the molecule were systematically replaced by a variety of ⁇ -amino acid residues. From these studies it followed that activity is highly dependent on the hydrophobic nature of the amino acid on the positions of the VaI and Leu residues. Furthermore, the Pro residue can be replaced with GIy or Leu without affecting the antibacterial activity. Finally, replacement of the D Phe residue by other amino acids is allowed, provided that the side chains in these are bulky and of a hydrophobic nature. Grotenbreg et al. focused on the design of GS analogs in which one or both ⁇ -turn regions were varied.
  • the D Phe or Pro residues were modified by attachment of different groups to the phenyl or pyrrolidine ring.
  • the D Phe-Pro motif was replaced with various sugar amino acids.
  • modification of the ⁇ -turn did not result in peptides with an improved biological profile.
  • Antibacterial and hemolytic activity turned out to correlate: whenever an analog with decreased hemolytic activity was identified, this analog also showed decreased antibacterial activity and vice versa.
  • the group of Izumiya has synthesized some GS analogs in which either the number of charges or the nature of the hydrophobic residues was altered. Unfortunately, these molecules were evaluated for their antibacterial activity only and thus nothing can be said about the relationship, if any, between peptide hydrophobicity and hemolytic properties. Hodges and co-workers prepared various analogs of GS by systematically replacing an amino acid by its enantiomer, using either GS or ring homologs as starting point. Some of the extended GS analogs, having four positive charges, possess altered amphiphilic properties and were found to possess significantly less hemolytic activity while retaining, to a certain extent, their antibacterial activity.
  • the present invention is based on work carried out by the present inventors to develop GS derivatives, which display a better therapeutic index than GS itself.
  • the inventors observed that increasing hydrophobicity is not itself sufficient, but rather that this should be counterbalanced by an increase in overall positive charge.
  • a gramicidin S derivative which displays an increased therapeutic index compared to gramicidin S, wherein said derivative has an increased hydrophobicity and increased overall positive charge in comparison to gramicidin S.
  • GS is a 10-mer cyclic peptide comprising two anti-parallel ⁇ -sheets bounded by two turn regions (see Figure 1).
  • the cyclic peptides of the present invention are based on this structure and generally conform to the structure as depicted by Formula (I) below:
  • R 1 -R 10 represent amino acids which may be substituted or possess various hydrophobic or charged side-chains and wherein the hydrophobicity and overall charge of the cyclic peptide is greater than GS.
  • Formula (I) represents a 10-mer cyclic peptide and whilst these are likely to be preferred, it will be appreciated that larger cyclic peptides may also be of use, such as 11 , 12, 13, 14, 15, 16 or even greater in length cyclic peptides.
  • Ri - R 10 of Formula (I) may all be different amino acids, or some amino acids may be identical.
  • the cyclic peptide conforms to the structure represented by general Formula (II) show below:
  • Ri - R 5 represent amino acids which may be substituted and wherein the overall charge and hydrophobicity of the cyclic peptide is greater than GS.
  • the cyclic peptide confirming to Formula (II) may be greater in length than as represented by the general Formula (II).
  • cyclic peptides of the present invention cannot be identical to GS itself. It should also be appreciated that the present invention is not to be limited in terms of particular enantiomeric forms of amino acids and all sterioisomers may be envisaged provided that the cyclic peptide structure can be formed.
  • R groups have the definitions as defined above for Formula (II) and wherein R 6 is a hydrophobic group as defined above in relation to R 3 , and is the same or different from the group found in the R 3 position .
  • R groups have the definitions as defined above for Formula (II) and wherein R 7 is adamantyl -2-glycine or adamantyl-2-alanine
  • Examples of larger 14 amino acid structures in accordance with the present invention include the structures which conform to the structure represented by general formula (Vl) shown below:
  • R groups have the same definitions as defined above for formula (II), wherein each R 3 group is independently selected and may be the same or different to one or more other R 3 groups.
  • each R 1 group may be selected independently, each R 2 group may be selected independently and each R 5 group may be selected independently.
  • both R 1 groups may be the same, each of the R 2 groups may be the same and each R 5 group may be the same.
  • the cyclic peptide of the present invention conforms to Formula II, wherein
  • R 1 is a substituted or unsubstituted proline (Pro);
  • R 2 is an amino acid having a positively charged side chain
  • R 3 is an amino acid having a hydrophobic group
  • R 4 is an amino acid having a positively charged side chain
  • R 5 is substituted or unsubstituted D-phenylalanine (D-Phe).
  • R 1 and/or R 5 may independently be a positively charged side chain, a hydrophobic group or any other suitable group.
  • R 2 and R 4 may be the same or different.
  • Positively charged side chains as used herein throughout are understood to include a group, such as a free amino group which may be protonated under physiological conditions so as to provide a positive charge.
  • the R 2 and R 4 groups may be amino acids which naturally possess said positively charged side groups, such as arginine, lysine and ornithine, or they may be amino acids which are substituted by addition of a suitable positively charged side group.
  • R 2 and R 4 are both ornithine (Om), lysine (Lys), arginine (Arg) or another amine containing functionality that holds a positive charge under physiological conditions.
  • R 3 is adamantyl-L-glycine or adamantyl-L-alanine; valine, alanine, or glycine substituted with a C 6 - Ci 4 alkyl group (such as dodecanoyl), or a cyclic alkyl group, as well as alkylated adamantane groups, isomers of adamantanes and di- or triadamantane groups, and tert-butyl groups or alkyl chains.
  • R 3 can also be the corresponding omega-amino acid.
  • cyclic peptide of the present invention is: Pro Orn R 11 Om D-Phe
  • both R 11 groU ps are the same and are adamantyl-L-glycine or adamantyl- L-alanine, or each R 11 .is different and one R 11 is adamantyl -L-alanine and the other is ⁇ -tert-buty!-L-alanine, or one Ri 1 is valine and the other is L-2- aminododecanoic acid.
  • R 1 /R 5 turn regions may be replaced by a suitable group.
  • suitable group include, Gly/Pro turns, D-Tyr/Pro, other hydrophobic dipeptides, substituted omega-amino acids and sugar amino acids and derivatives.
  • the cyclic peptides of the present invention are intended to possess an increased therapeutic index in comparison to GS.
  • the therapeutic index is defined as hemolytic activity/MIC (where both values are defined in terms of the same concentration parameters, such as ⁇ g/ml) and wherein hemolytic activity is understood as the concentration of peptide needed to induce 50% hemolysis in a blood sample and the Minimum Inhibitory Concentration is the lowest concentration of the peptide that will inhibit the visible growth of a micro-organism after overnight incubation.
  • the cyclic peptides of the present invention may display poorer antimicrobial activity compared to GS, but significantly reduced hemolytic activity, or may show substantially the same or better antimicrobial activity as well as a decreased hemolytic activity.
  • antimicrobial should be taken to refer to any cyclic peptide exhibiting antimicrobial or antibiotic activity (biostatic or biocidal).
  • antimicrobial peptides may exhibit antiviral, antifungal and/or antibacterial activities and in a preferred embodiment, the antimicrobial peptides useful in the instant invention may be antibacterial.
  • the cyclic peptides of the present invention display antimicrobial activity against Gram positive and/or Gram negative bacteria, especially both.
  • the cyclic peptides of the present invention display an efficacious antimicrobial activity against MRSA, VRE and/or other highly resistant strains of bacteria.
  • the cyclic peptides of the present invention display broad spectrum activity against a variety of microorganisms, especially bacteria.
  • the cyclic peptides of the present invention may also be complexed, combined or otherwise associated with other molecules which serve to enhance the efficacy of the peptides.
  • the cyclic peptides may be complexed with cyclodextrin so as to reduce toxicity, if required, ((a) Salem II, Duzgunes N Int. J. Pharmaceut. 2003, 250, 403-414. (b) CaI K, Centkowska K, Eur. J. Pharm. Biopharm. 2008, 68, 467-478. (c) Uekama K. Chem. Pharm. Bull. 2004, 52, 900- 915. (d) Szejtli J Chem. Rev. 1998, 98, 1743-53).
  • the present invention has been exemplified in relation to the generation of GS derivatives which display an increased therapeutic index in comparison to GS, the present invention should not be construed as being only limited to the development of such GS derivatives.
  • the observations by the present inventors in terms of increasing overall charge (especially positive charge) and hydrophobicity may be used to develop further cyclic peptide derivatives based on different cyclic peptide "parent" molecules.
  • a method of providing a derivative antimicrobial cyclic peptide having an increased therapeutic index in comparison to a parent cyclic peptide comprising modifying a parent cyclic peptide so as to increase overall charge and hydrophobicity so as to provide a derivative cyclic peptide and identifying whether or not said derivative cyclic peptide displays an increased therapeutic index in comparison to the parent cyclic peptide, from which it is derived.
  • the derivative cyclic peptide is derived from a parent cyclic peptide by certain defined amino acid substitutions with respect to the parent cyclic peptide.
  • the substituted amino acids may be conventional amino acids, or amino acids which have been modified so as to comprise appropriate charged side chains, hydrophobic groups or be able to form a suitable turn structure in the peptide. Exemplary groups have been described in detail hereinabove.
  • US6358921 discloses a number of 14-mer GS derivatives. Such derivatives may be used as "parent" cyclic peptides in accordance with the above method and further derivatized by way of increasing overall change and hydrophobicity. Another parent peptide is a cyclic decamer that contains four omega-amino acids rather than alpha-amino acids. Examples of such further derivatives are shown below:
  • AdaAla 2x Orn replaced by AdaGly/1 x
  • AdaAla 2 x Orn replaced by AdaGly/2 x
  • the present invention further provides a pharmaceutical composition comprising an antimicrobial peptide as described herein, together with a pharmaceutically acceptable carrier therefore.
  • compositions provided by this invention may comprise an antimicrobial peptide optionally in combination with a further therapeutic agent in a single preparation/formulation such that when the further therapeutic agent is present, the two active ingredients are administered to a subject together and at the same time.
  • the medicament or treatment may comprise two or more different preparations/formulations each containing either one or more additional therapeutic agents and/or one or more antimicrobial peptides.
  • the further agent(s) may be administered together with and at the same time as, an antimicrobial peptide or, alternatively, separately from (i.e. before or after) the antimicrobial peptide.
  • the treatment of an infection comprises the administration or use of one or more different compositions each containing one or more additional therapeutic agent(s) and/or one or more antimicrobial peptides
  • the additional therapeutic agent(s) may be administered topically and/or orally concurrently with or separately from an antimicrobial peptide which may be administered topically and/or parenterally, or indeed vice versa.
  • compositions of this invention may be formulated for topical administration and as such may be present as an ointment, solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water liquid emulsion.
  • Such formulations may be particularly useful where the microbial infection affects, for example, the skin.
  • antimicrobial peptides described above may be administered in a continuous and/or delayed fashion, via some form of delivery device.
  • Such devices are advantageous, particularly for the administration of antifungal compositions as they may allow a prolonged period of treatment relative to for example, an oral or intravenous medicament.
  • topical delivery devices may include, for example, a patch, dressing, bandage or plaster adapted to release a compound or substance to the skin or wound of a a patient.
  • a person of skill in the art would be familiar with the materials and techniques which may be used to deliver a compound or substance in such a manner.
  • the antimicrobial peptide(s) for use in the present invention may be combined with some form of matrix or substrate, such as a nonaqueous polymeric carrier, to render it suitable for use in a delivery system.
  • the antimicrobial peptide/matrix or substrate mixture may be further strengthened by the use of, for example, a woven or knot, non-woven, relatively open mesh fabric, to produce a patch, bandage, plaster or the like which may be releasably attached to a particular region of a patient's body. In this way, while in contact with a patient's skin, the delivery device releases the antimicrobial peptide to a wound or to the skin.
  • the peptides of the present invention may be prepared by conventional solid phase synthesis methods (Grotenbreg GM, Kronemeijer M, Timmer MSM, El Oualid F, van Well RM, Verdoes M, Spalburg E, van Hooft PAV, de Neeling AJ, Noort D, van Boom JH, van der Marel GA, Overkleeft HS, Overhand M "A Practical Synthesis of Gramicidin S and Sugar Amino Acid Containing Analogues" J. Org. Chem. 2004, 69, 7851) known in the art, followed by cleavage from the resin, cyclisation and deprotection, as discussed generally in the detailed description herein below.
  • the present invention provides use of antimicrobial cyclic peptides as described herein for the manufacture of a medicament for treating a microbial infection.
  • antimicrobial cyclic peptide as described herein for use in treating a microbial infection.
  • a method of treating a microbial infection of a subject comprising the step of: administering an effective amount of an antimicrobial cyclic peptide in accordance with the present invention to a subject in need thereof, to treat the nicrobial infection of said subject.
  • the compounds of the invention may also be administered in conjunction/combination with other treatments.
  • the patient is typically an animal, e.g a mammal, especially a human.
  • )hysiologically acceptable salt, ester or other physiologically functional derivative hereof described herein may be presented as a pharmaceutical formulation, iomprising the compound or physiologically acceptable salt, ester or other ihysiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic nd/or prophylactic ingredients.
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation.
  • the formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • compositions suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent.
  • Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored.
  • Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner.
  • Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope.
  • An active compound may also be formulated as dispersable granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
  • Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water liquid emulsion.
  • Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use. .
  • Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
  • compositions suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
  • solutions may be prepared and stored in a ready to use condition, (e.g. without the need for further formulation such as dilution into a useable concentration), in light-excluding containers such as sealed bottles, ampoules, blister packages and the like. Such containers are preferably provided in a sterile condition.
  • Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi- dose containers which are sealed after introduction of the formulation until required for use.
  • an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
  • An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly.
  • Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion- exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
  • Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
  • such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.
  • suitable liquid propellants include propane and the chlorofluorocarbons
  • suitable gaseous propellants include carbon dioxide.
  • Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.
  • Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
  • an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
  • Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
  • the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl Dleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions D ⁇ suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
  • a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
  • Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer.
  • a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkled with the formulation and then applied to the site to be treated.
  • Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form.
  • conventional water soluble excipients such as lactose or sucrose, may be incorporated in the powders to improve their physical properties.
  • suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients.
  • These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.
  • Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include a veterinarily acceptable water-miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • a veterinarily acceptable water-miscible solvent for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • the liquid concentrates may be administered to the drinking water of animals.
  • Figure 1 shows the structure of GS
  • Figure 2 shows hemolytic activity of GS analogs 1-11 on human red blood cells. Experiments were carried out in triplicate, a) Peptides 1-7 compared to GS. b) Inverted GS analogs 8-11 compared to GS. Compound 5 did not dissolve well in the buffers used for the assay and was therefore excluded.
  • Figure 3 shows a Comparison of 1 H NMR spectra for compounds 9 and 11 in the range 9-7.5 ppm. a) The 1 H NMR spectrum of 9 in the absence (left) and presence (right) of TFA. b) Convergence to one major conformer is seen when peptide 9 is heated to 333K. The NH signals of the major symmetrical conformer are indicated by arrows, c) Heating of 11 does not have any effect on the ratio between the conformers. All spectra were recorded in CD 3 OH at 600 MHz.
  • Figure 4 shows Hemolytic activity of GS analogs 12-19, 23 and 24
  • Reactions were monitored by thin layer chromatography on aluminum coated silica sheets (Merck, silica 60 F 254 ), using visualization by spraying with a solution of 25 g (NH 4 ) 2 MoO 4 , 10 g (NH 4 ) 4 Ce(SO 4 ) 4 in 100 ml H 2 SO 4 and 900 ml H 2 O, or a solution of 20% H 2 SO 4 in ethanol, followed by charring at ⁇ 150°C.
  • Column chromatography was carried out with silica gel (Screening Devices bv, 40-63 ⁇ m particle size, 60 A), using undistilled solvents.
  • NMR spectra were recorded on a Bruker AV400 or a Bruker DMX600 using deuterated solvents.
  • Fmoc- ⁇ (cyclohexyl)-L-glycine Fmoc-Chg-OH was purchased from Chemlmpex.
  • Fmoc- ⁇ -cyclohexyl-L-alanine Fmoc-Cha-OH
  • Fmoc-L- ⁇ -t-butylglycine Fmoc-tBuGly-OH
  • Fmoc-tBuGly-OH Fmoc-tBuGly-OH
  • LC-MS analysis was performed on a Jasco HPLC-system (detection simultaneously at 214 and 254 nm) coupled to a Perkin Elmer Sciex API 165 mass instrument with a custom-made Electronspray Interface (ESI).
  • An analytical Gemini C t8 column (Phenomenex, 50 x 4.60 mm, 3 micron) was used in combination with buffers A: H2O, B: MeCN and C: 1.0% aq. TFA.
  • An analytical Alltima CN column (Alltech, 150 x 4.6 mm, 5 micron) was used for analysis of cyclised protected peptides and peptide 5.
  • a Finnigan Surveyor HPLC system with a Gemini C18 50 * 4.6 mm column (Phenomenex, Torrance, CA, USA) (detection at 200-600 nm), coupled to a Thermo Finnigan LCQ Advantage Max mass spectrometer (Breda, The Netherlands) with electrospray ionization (ESI; system 1) was used, with the same buffers as described above.
  • ESI electrospray ionization
  • RP-HPLC purification of the peptides a Gilson GX-281 automated HPLC system (Gilson), supplied with a preparative Gemini Ci 8 column (Phenomenex, 150 x 21.2 mm, 5 micron) with buffers A: 0.1% aq.
  • TFA and B MeCN, or a BioCAD 'Vision' automated HPLC system (PerSeptiveBiosystems, inc.) supplied with a preparative Gemini Ci 8 column (Phenomenex, 150 x 21.2 mm, 5 micron) were used.
  • a preparative Alltima CN column Alltech, 150 x 22 mm, 5 micron
  • the applied buffers were A: H2O, B: MeCN and C: 1.0% aq. TFA.
  • A: H2O, B: MeCN and C 1.0% aq. TFA.
  • residue numbering was used as depicted on the right. All GS analogs were assigned as much as possible.
  • GS and its analogs were synthesized as reported previously.
  • the 'inverted' GS analogs 8-11 were synthesized starting from highly acid-labile HMPB-MBHA resin.
  • MBHA resin (Novabiochem, MBHA resin HLHCI 1 100-200 Mesh, theoretical load: 1.2 mmol g " ⁇ 2 g) was coupled to HMPB linker as described hereafter.
  • the resin needed for preparation of 5 was prepared from HMPB-MBHA resin, which was loaded with Fmoc-AdaAla-OH as described above.
  • Peptide synthesis was performed using standard Fmoc SPPS methods, either manually or in an automated fashion.
  • Peptides 6-7 were synthesized in the same way, but with Fmoc- Val-OH and Fmoc-Leu-OH replaced by Fmoc-Chg-OH and Fmoc-tBuGly-OH or Fmoc-Cha-OH and Fmoc-tBuAla-OH, respectively.
  • Fmoc-Adamantylglycine and Fmoc-Adamantylalanine were coupled with 90% HATU coupling reagent with respect to 1.5 equivalent amino acid.
  • Automated SPPS was executed with preloaded Fmoc-Orn(Boc)-HMPB- MBHA resin in the following order: Fmoc-Leu-OH/Fmoc-AdaAla-OH, Fmoc- Orn(Boc)-OH, Fmoc-Val-OH/Fmoc-AdaGly-OH, Fmoc-Orn(Boc)-OH, Fmoc- D Phe- OH, Fmoc-Pro-OH, Fmoc-Orn(Boc)-OH, Fmoc-Leu-OH/Fmoc-AdaAla-OH, Fmoc- Orn(Boc)-OH, Fmoc-Val-OH/Fmoc-AdaGly-OH,
  • the crude linear precursor of the peptide (150 ⁇ mol) was taken up in DMF (7.5 ml) and added dropwise over 1h to a solution of PyBOP (0.39 g, 750 ⁇ mol, 5 eq), HOBt (0.10 g, 750 ⁇ mol, 5 eq) and DIPEA (0.39 mi, 2.25 mmol, 15 eq) in DMF (120 ml). After stirring overnight, the solvent was evaporated and the residue applied to a LH-20 size-exclusion column, using MeOH as eluens. The peptide was obtained as a yellowish solid.
  • the crude protected peptide (216 mg) was dissolved in DCM (11 ml) and cooled to 0 ° C. TFA (11 ml) was added slowly and the mixture warmed to rt. After stirring for 30 min, solvents were evaporated and the residue coevaporated with toluene (three times) to remove all traces of TFA.
  • the crude peptide was obtained as a yellowish solid and purified by RP-HPLC, using gradients of H 2 O/MeCN/1 % TFA. The fractions containing the pure product were pooled, concentrated and freeze-dried to yield the pure peptides.
  • the following bacterial strains were used: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and the MRSA strains MRSA-NT 1110301981 H-tO34-PVL+, MRSA-NT N133-tO34-PVL-, MRSA-NT N229-tO34- PVL- and MRSA-Cluster218 USA300-1110301146 PVL+.
  • Freshly drawn heparinized blood was centrifuged for 10 minutes at 1000g at 1O 0 C. Subsequently, the erythrocyte pellet was washed three times with 0.85% saline solution and diluted with saline to a 1/25 packed volume of red blood cells.
  • the peptides to be evaluated were dissolved in a 30% DMSO/0.5 mM saline solution to give a 1.5 mM solution of peptide. If a suspension was formed, the suspension was sonicated for a few seconds. A 1% Triton-X solution was prepared.
  • the plates were centrifuged at 1000g at 1O 0 C for 4 min.
  • 50 ⁇ l of the supernatant of each well was dispensed into a corresponding well.
  • the absorbance at 405 nm was measured and the percentage of hemolysis was determined.
  • the JNH-H CI coupling constants were similar to those observed in GS; for D-phenylalanine a couping constant of ⁇ 4 Hz was observed; for the other residues JNH-HQ values were between 7 and 9 Hz.
  • the JNH-H O coupling constants of the unsymmetrical conformer of 9 showed a completely different picture: one D-phenylalanine had a JNH-H O value of 5.2 Hz and the other D Phe was not observed, while one of the ornithine NH signals showed a remarkably low coupling constant of 3.4 Hz. Also, both leucines displayed lowered JNH-H O values of -6.8 Hz.
  • IR (pure): 3405.3; 3270.2; 2959.2; 2927.9; 2904.3; 2340.1; 2359.6; 1683.7; 1682.9; 1674.4; 1668.5; 1661.7; 1659.2; 1651.7; 1644.8; 1639.7; 1634.0; 1628.2; 1622.4; 1616.0; 1575.0; 1568.3; 1563.7; 1553.7; 1557.7; 1549.3; 1538.2; 1532.0; 1526.7; 1520.5; 1516.0; 1505.9; 1471.3; 1463.3; 1455.7; 1452.2; 1447.8; 1435.9; 1344.0; 1200.5; 1134.0; 839.0; 800.2; 748.2; 722.3; 701.8; 667.9; 506.4.
  • IR (pure): 3278.0; 3059.7; 2902.3; 2848.6; 1683.6; 1674.0; 1667.7; 1656.4; 1651.6; 1639.7; 1634.1 ; 1557.6; 1553.1 ; 1543.8; 1537.9; 1531.8; 1526.0; 1506.1 ; 1450.4; 1436.1 ; 1344.5; 1312.4; 1201.6; 1181.7; 1135.8; 834.4; 799.2; 747.7; 722.0; 701.7; 667.8; 589.0; 507.5.
  • LC/MS retention time 12.57 min (10-90% MeCN, 15 min run).
  • IR (pure): 3286.0; 3064.7; 2957.4; 2874.8; 1699.6; 1694.1 ; 1687.8; 1683.7; 1674.0; 1667.9; 1661.2; 1657.2; 1651.6; 1639.8; 1634.4; 1562.7; 1557.7; 1553.6; 1548.0; 1543.8; 1538.2; 1531.8; 1525.8; 1520.1 ; 1516.0; 1505.9; 1496.2; 1479.5; 1475.9; 1471.5; 1463.3; 1455.6; 1435.9; 1417.9; 1398.4; 1367.7; 1201.6; 1183.2; 1135.5; 836.0; 799.7; 748.0; 722.2; 701.9; 667.9; 589.6; 558.2; 515.8.
  • IR (pure): 3266.0; 3066.8; 2925.8; 2852.4; 1699.7; 1687.9; 1683.6; 1674.0; 1668.0; 1661.5; 1657.5; 1651.5; 1639.5; 1634.0; 1557.7; 1553.6; 1547.7; 1543.7; 1538.1 ; 1532.1 ; 1526.4; 1520.3; 1505.8; 1455.3; 1448.6; 1435.9; 1339.6; 1201.9; 1184.6; 1135.4; 835.9; 799.8; 748.0; 722.2; 701.9; 667.9; 589.9; 559.4; 507.7.
  • IR (pure): 3271.9; 3063.6; 2969.8; 1699.6; 1695.2; 1687.9; 1683.7; 1679.8; 1673.9; 1668.2; 1661.8; 1658.6; 16518; 1645.4; 1639.8; 1634.8; 1627.9; 1622.4; 1616.2; 1557.8; 1553.8; 1538.8; 1532.3; 1525.8; 1521.3; 1516.1 ; 1506.0; 1471.6; 1455.5; 1435.7; 1200.1 ; 1173.5; 1133.1 ; 873.3; 834.6; 798.8; 746.7; 722.0; 703.2; 667.9; 611.9; 605.4; 596.0; 590.1; 518.1; 507.6.
  • IR (pure): 3281.4; 3055.3; 2918.3; 2853.4; 1705.8; 1701.6; 1696.0; 1685.7; 1676.0; 1670.7; 1664.0; 1659.6; 1654.0; 1648.0; 1636.7; 1630.4; 1624.8; 1618.8; 1560.0; 1555.7; 1551.7; 1545.9; 1541.8; 1534.0; 1528.6; 1522.2; 1518.0; 1513.2; 1508.2; 1501.9; 1498.2; 1457.9; 1449.8; 1438.0; 1430.6; 1426.0; 1420.4; 1342.6; 1313.7; 1200.4; 1176.5; 1132.6; 834.2; 798.9; 749.9; 721.9; 701.8; 594.1; 504.7.
  • IR (pure): 3053.5; 2912.4; 2901.8; 2853.4; 2849.8; 2846.3; 1704.1; 1699.5; 1694.1 ; 1688.2; 1683.6; 1679.7; 1673.8; 1668.0; 1661.7; 1651.7; 1645.7; 1640.2; 1634.2; 1627.9; 1622.1 ; 1615.9; 1574.2; 1568.1 ; 1563.3; 1557.7; 1553.8; 1549.1 ; 1543.8; 1538.2; 1531.8; 1525.9; 1520.1 ; 1515.6; 1505.7; 1495.9; 1471.2; 1463.1 ; 1455.6; 1447.6; 1435.6; 1201.3; 1186.2; 1179.1 ; 1133.7; 837.8; 799.5; 722.2; 702.1 ; 595.9; 518.0.
  • Adamantaneacetic acid 72 (9.71 g, 50 mmol) was treated in the same way as adamantane-1-carboxylic acid 64.
  • the pure 1-Hydroxyethyladamantane was obtained as a white solid (8.53 g, 47.3 mmol, 95%).
  • the alcohol was subjected to a Swern oxidation in the same way as 65 to yield aldehyde 73 as a pale yellow oil (3.18 g, 17.9 mmol, 89%).
  • the product was immediately used for the next step.
  • Aldehyde 73 (3.46 g, 19.4 mmol) was treated in the same way as adamantane-1 -carboxaldehyde 66 to yield 74 as a white powder (0.57 g, 1.77 mmol, 9%).
  • R-(-)-phenylglycinol was dissolved in methanol (44 ml), activated molsieves (3 A) were added and the solution cooled to O 0 C.
  • a solution of aldehyde 73 (1.59 g, 8.95 mmol) in methanol (22 ml) was added dropwise and the reaction mixture stirred for 2 h at room temperature.
  • TMSCN was added dropwise and the resulting mixture stirred at room temperature for 16 h.
  • EtOAc was added and the reaction mixture stirred for 15 min. The layers were separated, the aqueous layer extracted with EtOAc and the combined organic layers washed with sat.
  • adamantyl-amino acids 69 and 77 were chosen because adamantane moieties are hydrophobic and thought to be ideally suited for interaction with lipid bilayers.
  • Adamantyl-L-glycine is selected as replacement for valine residues, and adamantyl-L-alanine serves as a replacement for leucine residues.
  • GS analogs 1-11 were designed, in which either one or more of the hydrophobic VaI and Leu residues were replaced by even more hydrophobic moieties (peptides 1-7), or the hydrophobic residues were swapped for cationic residues and vice versa (peptides 8-11, for similar approaches, see ref. 14b-e).
  • the first series of modifications would result in peptides more hydrophobic than GS, the second in peptides more hydrophilic than GS. In all cases, the turn regions were as in the parent compound, GS.
  • Fmoc-adamantyl-L-glycine 69 was synthesized according to a route published by Augeri and co-workers (Scheme 1 ).
  • Reagents and conditions a) LiAIH 4 , THF, 0 0 C to rt, 2 h, 97%. b) (CICO) 2 , DMSO, CH 2 CI 2 , -78 0 C, 94%. c) (R)-(-)-2-phenylglycinol, NaHSO 3 , KCN, H 2 0/Me0H 1 :1, rt, 2 h, then reflux, 16 h, 82%. d) 12 M HCI, HOAc, 80 0 C, 16 h, 74%. e) 20% Pd(OH) 2 , H 2 , MeOH/HOAc 5:1 , 18 h.
  • nucleophilic attack should take place antiperiplanar to the ⁇ -phenylgroup.
  • the energy of the transition state is lowered when the phenylgroup is perpendicular to the imine, leading to transition states A and B.
  • attack of the cyanide can take place from two sides.
  • A is then more favorable than B because of steric considerations, but also because this transition state is additionally stabilized by intramolecular hydrogen bonding.
  • B the hydroxymethyl group is close to both the incoming cyanide and the adamantylgroup and therefore this transition state is not favored. This would explain the observed stereoselectivity.
  • Reagents and conditions a) L LiAIH 4 , THF, 0 0 C to rt, 2 h, 95%. ii. (CICO) 2 , DMSO, CH 2 CI 2 , -78 0 C, 85% over two steps, b) (R)-(-)-2-phenylglycinol, NaHSO 3 , KCN, H 2 O/MeOH 1 :1 , rt, 2 h, then reflux, 16 h, 9%. c) (R)-(-)-2-phenylglycinol, activated molsieves 3 A, O 0 C to rt, 3 h.
  • decameric peptides were stepwise assembled on the solid support, with either leucine (1, 2), adamantyl-L-alanine (3-5), terf-butyl-L-alanine (6); cyclohexyl-L-alanine (7) or ornithine (8-11) being the first amino acid immobilized to the resin.
  • leucine (1, 2), adamantyl-L-alanine (3-5), terf-butyl-L-alanine (6); cyclohexyl-L-alanine (7) or ornithine (8-11) being the first amino acid immobilized to the resin.
  • the elongation towards the immobilized decapeptides, cleavage from resin, cyclization, deprotection and purification of the peptides proceeded uneventfully and the target peptides were obtained in high purity (>95 %) and in good yields.
  • the JiMH- H a values of the D-phenylalanine residues were in the range of 2-4 Hz 1 which is typical for an amino acid as part of a ⁇ -turn.
  • the NOE signals between the backbone amide NH protons were similar to the corresponding NOE crosspeaks as observed for GS.
  • the NMR data of these compounds strongly indicate a rigid cyclic beta-hairpin secondary structure in solution, which is also observed for GS.
  • GS shows, as expected, strong activity against Gram-positive bacteria and, to a lesser extent, Gram-negative bacteria. It is very effective against the MRSA strains as well.
  • Compounds 1 and 3 are as active as GS against Gram- positive bacteria and less active against Gram-negative bacteria, but 2 and 4 show less activity against all bacteria assayed.
  • Peptide 5 has completely lost its activity.
  • Peptide 6 shows a slight increase in selectivity against Gram-positive bacteria, and performs well against the MRSA strains.
  • Peptide 7 is less active against all bacteria.
  • 8 and 9 do not display any inhibition in bacterial growth, but peptides 10 and 11 are as active as GS towards Gram-positive bacteria. Furthermore, they show an increased activity against the two Gram-negative bacteria. The activity against the MRSA strains is comparable with that of GS.
  • peptides 12-18 and 24 hardly show any activity against the bacterial strains assayed. In contrast, peptides 19 and 23 perform as well as GS, also against the MRSA strains.
  • analogs 45-48 perform as well as or slightly better than GS, while peptides 40 and 51 barely show any activity. The other peptides assayed display less antibacterial activity than GS.
  • a frequently used procedure to assess the toxicity of a compound is by measuring its hemolytic activity at various concentrations.
  • the ability of the peptides 1-11 to lyse human red blood cells was determined.
  • the results are depicted in Figure 3.
  • GS shows high hemolytic activity.
  • Peptides 1-7 have hemolytic activity in the same range as GS, whereas peptide 8-11 are hardly active (11 and especially 10) or completely inactive (8, 9) at the final concentrations tested.
  • the same assay was performed for peptides 12-19, 23 and 24.
  • the majority of the compounds displays no hemolytic activity against red blood cells in the concentration range tested.
  • Peptides 13,19 and 23 are less hemolytic than GS.
  • Table 5 shows the therapeutic index of some of the cyclic peptides of the present invention derived from the hemolytic value and the antimicrobial activity in terms of the minimal inhibitory concentration.
  • MIC Minimal Inhibitory Concentration
  • Bold indicates better antibacterial activity for G- bacteria than GS itself.
  • the therapeutic index is defined as hemolytic activity/antimicrobial activity, where both values are in ⁇ g/ml.
  • MIC-values above 64 ⁇ g/ml a value of 128 ⁇ g/ml was used for the calculation, and for hemolysis values above 500 ⁇ g/ml, a value of 1000 ⁇ g/ml was used.
  • the hemolysis values are the results of three independent experiments and indicate the concentration of peptide needed ( ⁇ M) to induce 50% hemolysis. These values were calculated from the data shown in Figure 3.
  • Table 6 shows the therapeutic index of compounds 10 and 11 in terms of certain MRSA strains.
  • Table 7 LC/MS retention times and the number of charges of GS and analogs 1- 11 , ranked from least to most hydrophobic. LC-MS spectra were recorded using a gradient of 10- ⁇ 90% MeCN, and a run duration of 15 min.
  • the hemolytic activities of the analogs can be correlated to hydrophobicity as well: peptides more hydrophobic than GS show similar ability to lyse human red blood cells. Again, of the more hydrophilic compounds 11 and especially 10 show reduced hemolytic activity, while 8 and 9 have no activity at all.
  • composition of bacterial cell membranes differs from those of mammalian membranes.
  • Bacterial cell membranes contain a high percentage of the negatively charged phospholipids phosphatidylglycerol, cardiolipin and phosphatidylserine.
  • Mammalian cell membranes in contrast contain more zwitterionic and uncharged molecules such as phosphatidylethanolamine, phosphatidylcholine and cholesterol. The different charge distribution in the membranes may be influential for the partition of GS and its analogs into these.
  • peptides 10, 11, 19, and 23 Based on their decreased hemolytic activity combined with enhanced antimicrobial activity and their action on a broader spectrum of (Gram negative) bacteria as compared to GS, as well as their activity against MRSA strains, peptides 10, 11, 19, and 23 at least are interesting lead structures for the development of novel broad spectrum antibiotics against a wide spectrum of bacteria, possibly including multi-drug resistant strains.

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Abstract

La présente invention concerne des peptides cycliques antimicrobiens, qui présentent une activité hémolytique réduite et éventuellement une activité antimicrobienne comparativement aux peptides cycliques antimicrobiens parents dont ils dérivent. Selon un mode de réalisation, le peptide cyclique parent est la gramicidine S. L'invention concerne également un procédé permettant d'obtenir de nouveaux peptides cycliques antimicrobiens dérivés, qui ont été développés à partir d'un peptide cyclique antimicrobien parent et qui présentent une activité hémolytique réduite et/ou une activité antimicrobienne comparativement à la molécule parente dont ils dérivent.
PCT/IB2010/001250 2009-04-03 2010-04-06 Peptides cycliques antimicrobiens WO2010113042A1 (fr)

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RU2794363C1 (ru) * 2021-11-15 2023-04-17 Общество С Ограниченной Ответственностью "Валента-Интеллект" Фармацевтические композиции для лечения инфекционно-воспалительных заболеваний
WO2023085973A1 (fr) * 2021-11-15 2023-05-19 Общество С Ограниченной Ответственностью "Валента-Интеллект" Compositions pharmaceutiques pour traiter des maladies infectieuses-inflammatoires

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