WO2009055779A1 - Procédé pour l'inversion d'une résistance bactérienne à des antibiotiques métallo-b-lactamase et à la vancomycine - Google Patents

Procédé pour l'inversion d'une résistance bactérienne à des antibiotiques métallo-b-lactamase et à la vancomycine Download PDF

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WO2009055779A1
WO2009055779A1 PCT/US2008/081306 US2008081306W WO2009055779A1 WO 2009055779 A1 WO2009055779 A1 WO 2009055779A1 US 2008081306 W US2008081306 W US 2008081306W WO 2009055779 A1 WO2009055779 A1 WO 2009055779A1
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antibiotic
resistance
ctc
vancomycin
bacteria
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PCT/US2008/081306
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English (en)
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David Gershon
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Redox Pharmaceutical Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • 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

  • ⁇ -lactam antibiotics consist of a variety of molecules the structure of which contains a ⁇ -lactam nucleus. Since the initial introduction of penicillin, this group of antibiotics which includes Penicillins, Cephalosporins, Carbapenems and Monobactams has been the most widely used in the treatment of bacterial infections.
  • ⁇ -lactam antibiotics are bactericidal as a result of their inhibition of the synthesis of bacterial cell wall peptidoglycans.
  • the structural similarity between ⁇ -lactam antibiotics and the terminal dipeptide of the bacterial wall peptidoglycan enables these antibiotics to bind to the active site of bacterial penicillin binding proteins (PBPs) which are necessary for normal cell wall development and thus inactivate them.
  • PBPs penicillin binding proteins
  • All the ⁇ -lactam antibiotics carry a lactam ring and are therefore susceptible to ⁇ -lactamases including metallo- ⁇ -lactamases produced by mutant bacterial strains.
  • Bacterial ⁇ -lactamases which cleave the amide bond of the ⁇ -lactam ring thus inactivating ⁇ -lactam antibiotics, are a major cause of antibiotic resistance 1
  • Class B ⁇ -lactamases are metalloenzymes (Metallo-Beta-Lactamases, referred to herein as MBL) requiring one or two zinc ions in their active site for their activity.
  • MBLs present a distinct threat, since, unlike most other lactamases which have limited ranges of activity, they can degrade ⁇ -lactam antibiotics of most classes. In recent years mutant bacterial strains carrying MBLs have become more prevalent and are rapidly posing an increasingly serious problem in the fight against bacterial infections 1i3 ⁇ 5 .
  • MBLs are divided into three subclasses, B1 , B2 and B3.
  • MBLs are either mono-zinc or di-zinc enzymes 1 .
  • the zinc ions are located in the active site and are essential for catalytic activity 6 ' 7 ' 8 .
  • Class B1 enzymes possess a zinc site with an HXHXD sequence.
  • class B2 enzymes the site has a NXHXD sequence whereas class B3 enzymes have a zinc site with an H(Q)XHXHD sequence.
  • H stands for histidine
  • X stands for any amino acid
  • N stands for asparagine
  • D stands for aspartic acid
  • Q stands for glutamine.
  • Zinc ion coordination requires essential histidines.
  • Vancomycin is an aminoglycoside antibiotic which has been widely used against bacterial strains resistant to other antibiotics. Vancomycin is often the only antibiotic effective against certain drug-resistant bacteria. Vancomycin acts at the same site as the ⁇ -lactam antibiotics but binds the terminal dipeptide of the bacterial wall peptidoglycan rather than the PBPs with which the ⁇ -lactam antibiotics interact. This binding to the terminal dipeptide prevents the formation of the peptidoglycan layer of the bacterial cell wall and renders Vancomycin bactericidal.
  • Vancomycin resistance was first reported in the late 1980s and has become a major threat in the past several years 9 . Vancomycin resistance, initially discovered in Enterococci, was found to be plasmid-mediated. This plasmid which carries an operon containing an array of genes required for antibiotic resistance 10 has transferred to other bacteria including gram positive Staphylococcus aureus 11 ' 12 and Streptomyces species 13 .
  • the resistance gene products are: VanS (a histidine protein kinase), VanR (a response regulator), VanX (a D,D-dipeptidase), VanY (a D,D-carboxypeptidase), VanH (a dehydrogenase), and VanA (a ligase).
  • VanS histidine kinase
  • VanS/VanR constitute this two component signal transduction system where the histidine kinase (VanS) auto-phosphorylates and then trans-phosphorylates the response regulator, VanR. Consequently, VanR acts as a transcriptional regulator which is important in imparting vancomycin resistance 14 .
  • VanX is a zinc-containing D-Alanine:D-Alanine dipeptidase that confers vancomycin resistance in Enterococci and Staphylococcus and other bacteria 10 .
  • VanY is a zinc-containing D,D-carboxy- dipeptidase which is also required for resistance to vancomycin 10 .
  • CTC-96 (DOXOVIRTM) belongs to a family of molecules possessing a small cobalt (III) Schiff base complex that has potent anti-viral and anti-inflammatory activities. Its structure and method of preparation are disclosed in US patent 5,756,491 , the contents of which are hereby incorporated by reference.
  • CTC-96 The mode of action of CTC-96 is based on its binding to the nitrogen of the imidazole ring of histidine residues in proteins 15 . It can thus disrupt the structure and function of Zn fingers that contain one or two histidines coordinated about a zinc ion in proteins 16 . It can also disrupt other Zn containing motifs in proteins. Moreover, it inhibits enzymes that contain histidine in their active site, such as, thermolysin and thrombin 17 and may effectively inhibit some other serine and cysteine proteases. The structures of four of these enzymes, VanS, VanX, VanY, and VanA are particularly relevant to CTC-96 mediated disruption of vancomycin resistance.
  • CTC-96 is effective in reversing drug resistance to gram negative and gram positive bacteria.
  • CTC-96 has a wide spectrum of bacterial targets.
  • CTC-96 is suitable for treatment of acute bacterial infections and is expected to alleviate the severe effects of infections caused by antibiotic-refractive bacterial strains. It is effective because it can engage several rather than single bacterial targets during the course of infections.
  • Fig. 1 is a graph of the inhibition of various strains of s. aureus at varying concentrations of CTC-96.
  • CTC-96 may be administered orally, by intramuscular or intravenous injection, by topical application to the skin or by use of a transdermal patch or nasal spray.
  • CTC-96 is administered generally within several hours and, preferably, about within about 3-4 hours of administration of the antibiotic.
  • the CTC-96 may be administered prior to, simultaneously with or after the administration of the antibiotic.
  • Most preferably, the CTC-96 is administered within about 5 to 60 minutes of the administration of the antibiotic.
  • the amount of CTC-96 administered will vary with the particular antibiotic being administered, but in all cases should be an anti-antibiotic resistance effective amount, i.e., an amount sufficient to reduce or reverse the anti-biotic resistance of the bacteria.
  • CTC-96 is effective in the reversal of bacterial metallo- ⁇ -lactamase (MBL) based resistance to ⁇ -lactam antibiotics.
  • MBLs are all zinc-containing enzymes in which the zinc is essential for the catalytic function of the enzyme.
  • the Zn ion is coordinatively held by essential histidines.
  • CTC-96 interacts with Zn coordinating histidines and causes the ejection of the Zn, thus inactivating the Zn bearing MBL and re-imparting antibiotic sensitivity 16 .
  • CTC-96 is effective in the reversal of bacterial vancomycin resistance.
  • Vancomycin resistance is imparted by an array of plasmid encoded genes for VanS (a histidine protein kinase), VanR (a response regulator), VanX (a D,D-dipeptidase), VanY (a D,D-carboxypeptidase), VanH (a dehydrogenase), and VanA (a ligase).
  • VanS a histidine protein kinase
  • VanR a response regulator
  • VanX a D,D-dipeptidase
  • VanY a D,D-carboxypeptidase
  • VanH a dehydrogenase
  • VanA a ligase
  • VanS is a histidine kinase with an essential histidine that is phosphorylated and is used for transphosphorylation of VanR. Histidine phosphorylation also occurs on the nitrogen of the imidazole group. CTC-96 interacts with nitrogen of the imidazole group of histidines 17 . Thus, CTC-96 hampers the phosphorylation of the histidine crucial for the auto-phosphorylation of the histidine kinase and thus abolishes the transphosphorylation of VanR. This interruption of signal transduction will thus reverse the resistance of the bacterium to vancomycin.
  • VanX is a zinc-containing dipeptidase which prevents the interaction of vancomycin with its dipeptide target thus permitting the synthesis of the bacterial cell wall.
  • the enzyme contains two zinc-coordinating histidines (his 116, his 184) in a catalytic site motif that is homologous to that of Bacillus derived thermolysin 18 . Since CTC-96 inhibits thermolysin 19 , CTC-96 is a potential inhibitor of this essential enzyme involved in vancomycin resistance.
  • VanY VanY is a zinc-containing D,D-carboxy-dipeptidase which is required for resistance to vancomycin 10 . There is a close homology between the zinc- binding motifs of Va nX and VanY. Thus VanY is also a target for inhibition by CTC-96 and reversal of vancomycin resistance.
  • VanB the gene for which is chromosomal, not plasmid-borne, produces lactate which is substituted for the second alanine in the terminal dipeptide of the bacterial wall peptidoglycan by VanA.
  • This D-alanyl-lactate which substitutes for the dipeptide has very reduced affinity for vancomycin resulting in antibiotic resistance 10 .
  • the ligase Van A contains an essential histidine in its active site. Van A is, therefore, a target for inhibition by CTC-96 resulting in reversal of vancomycin resistance.
  • CTC-96 can act against certain toxins and virulence factors that are produced by either drug-sensitive or drug-resistant bacterial strains regardless of sensitivity, thus further expanding its potential for use as an antibiotic-enhancing and bacterial virulence-reducing agent.
  • Bacillus anthracis Lethal Factor is a secreted zinc-binding protein which contains the His-Glu-X-X-His zinc binding motif typical of zinc-endopeptidases.
  • the two major toxins produced by B. anthracis, lethal factor (LF) and edema factor (EF), are lethal only if combined with the third factor, protective antigen (PA).
  • PA protective antigen
  • LF mitogen-activated protein kinase (MAPK) kinases
  • MAPK mitogen-activated protein kinase
  • MAPKKs mitogen-activated protein kinase kinases
  • LF is a prime target for inactivation by CTC-96 since it contains zinc in its catalytic domain that is tetrahedrally composed of a water molecule and two coordinating histidines (His 686 and His 690) in a HExxH motif typical of the thermolysin family 24 .
  • His 686 and His 690 two coordinating histidines
  • LF specifically cleaves the N-terminal ends of MAPKK.
  • CTC-96 inhibition of LF will prevent the interruption of this essential signaling pathway.
  • CTC-96 has been shown to inhibit LF in a Fluorescent plate based assay and an HPLC based assay 25 .
  • CTC-96 has an IC 5O value for LF between 0.5 to 1 ⁇ M. This sensitivity is considered in the range of therapeutically viable inhibitors of LF 25 .
  • Staphylococcus aureus ⁇ -toxin is a membrane damaging protein produced by most pathogenic stains of this species and is considered a major virulence factor 26 ' 27 .
  • This protein contains three histidines that are essential for pore formation in cell membranes. Mutation in these histidines abolishes (his 35) or drastically reduces (his 48 and his 144) the hemolytic activity of this toxin by preventing completion of pore formation in the membrane 26 . Interaction of CTC 96 with any or all of these histidines in this extra-cellular protein can abolish toxin activity and thus alleviate the toxic effect of the bacterial infection regardless of sensitivity or resistance to antibiotics.
  • Staphylococcus aureus produces a D-alanine:D-alanine liqase (DDL).
  • ZmpB and ZmpC are zinc-metalloproteinase virulence factors produced by a variety of pathogens like Streptomvces pneumoniae. These factors induce inflammation in the lower respiratory tract 28 ' 29 which entails an elaborate chain of reactions involving the release of heparin-binding epidermal growth factor receptor causing an induction of mucin overexpression, which in turn promotes lung infection by reducing airflow and by inhibiting antibacterial agents 30 . Both ZmpB and ZmpC have a zinc motif in their active site which contains the HEXXHE sequence rendering them targets for CTC-96 inhibition via binding to the histidines in this motif.
  • CTC-96 Since these metalloproteinases are either extracellular or surface proteins, they are inhibited by CTC-96 without its penetration into the bacterial cell. CTC-96 can, therefore, act against these virulence factors even in multiple drug-resistant (MDR) bacterial strains. MDR is based on the presence of an active cellular efflux pump against a variety of unrelated anti-bacterial agents. In those cases in which CTC-96 can act extra-cellularly it is unaffected by MDR. Thus, CTC-96 reduces the virulence and pathogenicity of many bacterial strains regardless of their degree of sensitivity or resistance to other antibiotics. This is a particularly important aspect of the anti-bacterial virulence mode of action of CTC-96.
  • MDR drug-resistant
  • Methicillin-resistant Staphylococcus aureus strains are resistant to all ⁇ -lactam antibiotics as a result of the presence of a penicillin binding protein MecA (aka: PBP2' and PBP2a MecA).
  • MecA competes with the normal cell wall synthesizing PBP that is inhibited by ⁇ -lactams thus allowing cell wall synthesis in the presence of ⁇ -lactams.
  • This Methicillin resistance is regulated by the chromosomal mec-divergon which encodes a signal-transduction system that includes the MecR1 and Med proteins and MecA, the product of the gene mecA, which exerts the resistance to ⁇ -lactams.
  • MecR1 is a sensor/transducer fusion protein which transcends the bacterial membrane and Med is a repressor of the mecA gene.
  • MecR1 has an extra-membrane penicillin binding domain which, upon ⁇ -lactam binding, transmits a signal through the membrane which triggers the activation of the metalloprotease moiety of the molecule found in the cytoplasmic side of the molecule 1 ' 2 ' 3 .
  • This Zn metalloprotease in turn cleaves Med , the repressor of the mecA gene, thus allowing MecA synthesis.
  • MecA mediates ⁇ -lactam resistance by the following mechanism.
  • MecA is a very low-affinity ⁇ -lactam binding protein, found only in gram-positive bacteria (e.g., Bacillus, Staphylococcus and Enterococci). This low-affinity of MecA for ⁇ -lactams allows cell wall synthesis at ⁇ -lactam concentrations that would be lethal with other higher affinity PBPs. Thus a crucial step in this resistance system is the Zn-metalloprotease mediated inactivation of the repressor of the mecA gene.
  • the metalloprotease moiety of the MecR1 protein contains a Zn ion which is coordinated by two essential histidines (his 204 and his 208 in the case of Staphylococcus aureus) 2 .
  • This feature makes the molecule a potential target for CTC- 96. Inhibition of this protease by CTC-96 is expected to render Methicillin-resistant strains of gram positive bacteria susceptible to ⁇ -lactamases.

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Abstract

L'invention concerne un procédé pour réduire ou inverser la résistance de bactéries à des antibiotiques par administration d'une quantité efficace de résistance anti-antibiotique de CTC-96 sur une durée relativement proche de la durée d'administration de l'antibiotique.
PCT/US2008/081306 2007-10-26 2008-10-27 Procédé pour l'inversion d'une résistance bactérienne à des antibiotiques métallo-b-lactamase et à la vancomycine WO2009055779A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US98291207P 2007-10-26 2007-10-26
US60/982,912 2007-10-26
US10860908P 2008-10-27 2008-10-27
US61/108,609 2008-10-27

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WO2009055779A1 true WO2009055779A1 (fr) 2009-04-30

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040224904A1 (en) * 2001-10-15 2004-11-11 Benedikt Sas Preparation and use of carbohydrate-based bicyclic ring structures with antimicrobial and cytostatic activity
US20070142348A1 (en) * 2003-06-20 2007-06-21 Redox Pharmaceutical Corporation, Audubon Biomedical Science And Technology Pack Microbicidal, prophylactic and therapeutic effect of ctc-96 on papilloma viruses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040224904A1 (en) * 2001-10-15 2004-11-11 Benedikt Sas Preparation and use of carbohydrate-based bicyclic ring structures with antimicrobial and cytostatic activity
US20070142348A1 (en) * 2003-06-20 2007-06-21 Redox Pharmaceutical Corporation, Audubon Biomedical Science And Technology Pack Microbicidal, prophylactic and therapeutic effect of ctc-96 on papilloma viruses

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