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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
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  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3513—Protein; Peptide

Definitions

• the present invention concerns novel drugs for use in combating infectious microorganisms, in particular bacteria. More particular the invention concerns 5 peptide nucleic acid (PNA) sequences, which are modified in order to obtain novel PNA molecules with enhanced anti-infective properties.
• PNA peptide nucleic acid
• 15 clinicians is more than 100.
• antibiotics are products of natural microbic populations and resistant traits found in these populations can disseminate between species and appear to have been acquired by pathogens under selective pressure from antibiotics used in 20 agriculture and medicine (Davis 1994).
• Antibiotic resistance may be generated in bacteria harbouring genes that encode enzymes that either chemically alter or degrade the antibiotics. Another possibility is that the bacteria encodes enzymes that makes the cell wall impervious to antibiotics or encode efflux pumps that eject antibiotics from the cells before they can exert their effects.
• Antisense agents offer a novel strategy in combating diseases, as well as opportunities to employ new chemical classes in the drug design.
• Oligonucleotides can interact with native DNA and RNA in several ways.
• duplex formation between an oligonucleotide and a single stranded nucleic acid Another is triplex formation between an oligonucleotide and double stranded DNA to form a triplex structure.
• PNA Peptide nucleic acids
• One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
• PNA hybridises with complementary nucleic acids through Watson and Crick base pairing and helix formation (Egholm et al. 1993
• PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes.
• the resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm's. This high thermal stability might be attributed to the lack of charge repulsion due to the neutral backbone in PNA.
• PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex, there is seen an 8 to 20°C drop in the Tm.
• homopyrimidine PNA oligomers form extremely stable PNA 2 -DNA triplexes with sequence complementary targets in DNA or RNA oligomers.
• PNA's may bind to double stranded DNA or RNA by helix invasion.
• PNA polyamide backbone (having appropriate nucleobases or other side chain groups attached thereto) is not recognised by either nucleases or proteases and are thus not cleaved.
• PNA's are resistant to degradation by enzymes unlike nucleic acids and peptides.
• target bound PNA can cause steric hindrance of DNA and RNA polymerases, reverse transcription, telomerase and the ribosome's (Hanvey et al. 1992 (33), Knudsen et a. 1996 (34), Good and Nielsen 1998 (39,40), etc.
• a general difficulty when using antisense agents is cell uptake.
• a variety of strategies to improve uptake can be envisioned and there are reports of improved uptake into eukaryotic cells using lipids (Lewis et al. 1996 (35)), encapsulation (Meyer et al. 1998 (36)) and carrier strategies (Nyce and Metzger 1997 (37), Pooga et al, 1998 (38)).
• WO 99/05302 discloses a PNA conjugate consisting of PNA and the transporter peptide transportan, which peptide may be used for transport cross a lipid membrane and for delivery of the PNA into interactive contact with intracellular polynucleotides.
• US-A-5 777 078 discloses a pore-forming compound which comprises a delivery agent recognising the target cell and being linked to a pore-forming agent, such as a bacterial exotoxin.
• the compound is administered together with a drug such as PNA.
• PNA may have unique advantages. It has been demonstrated that PNA based antisense agents for bacterial application can control cell growth and growth phenotypes when targeted to Escherichia coli rRNA and mRNA (Good and Nielsen 1998a,b (39,40) and WO 99/13893).
• US-A-5 834 430 discloses the use of potentiating agents, such as short cationic peptides in the potentiation of antibiotics. The agent and the antibiotic are co- administered.
• WO 96/11205 discloses PNA conjugates, wherein a conjugated moiety may be placed on terminal or non terminal parts of the backbone of PNA in order to functionalise the PNA.
• the conjugated moieties may be reporter enzymes or molecules, steroids, carbohydrate, terpenes, peptides, proteins, etc. It is suggested that the conjugates among other properties may possess improved transfer properties for crossing cellular membranes.
• WO 96/11205 does not disclose conjugates, which may cross bacterial membranes.
• WO 98/52614 discloses a method of enhancing transport over biological membranes, e.g. a bacterial cell wall.
• biological active agents such as PNA may be conjugated to a transporter polymer in order to enhance the transmembrane transport.
• the transporter polymer consists of 6-25 subunits; at least 50% of which contain a guanidino oramidino sidechain moiety and wherein at least 6 contiguous subunits contain guanidino and/or amidino sidechains.
• a preferred transporter polymer is a polypeptide containing 9 arginine.
• the present invention concerns a new strategy for combating bacteria. It has previously been shown that antisense PNA can inhibit growth of bacteria. However, due to a slow diffusion of the PNA over the bacterial cell wall a practical application of the PNA as an antibiotic has not been possible previously. According to the present invention, a practical application in tolerable concentration may be achieved by modifying the PNA by linking a peptide or peptide-like sequence, which enhances the activity of the PNA.
• the present invention concerns a modified PNA molecule of formula (I):
• Peptide is any amino acid sequence and
• PNA is a Peptide Nucleic Acid, and pharmaceutically acceptable salts thereof.
• the present invention concerns a modified PNA molecule of formula (I)
• Peptide is a cationic peptide or cationic peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the formula (II):
• A consists of from 1 to 8 non-charged amino acids and/or amino acid analogs
• B consists of from 1 to 3 positively charged amino acids and/or amino acid analogs; C consists of from 0 to 4 non-charged amino acids and/or amino acid analogs; D consists of from 0 to 3 positively charged amino acids and/or amino acid analogs; n is 1-10; and the total number of amino acids and/or amino acid analogs is from 3 to 20.
• the Peptide of the present invention contains from 2 to 60 amino acids.
• the amino acids can be negatively, non-charged or positively charged naturally occurring, rearranged or modified amino acids.
• the peptide contains from 2 to 18 amino acids, most preferred from 5 to 15 amino acids.
• a in formula (II) consists of from 1 to 6 non-charged amino acids and/or amino acid analogs and B consists of 1 or 2 positively charged amino acids and/or amino acid analogs.
• A consists of from 1 to 4 non-charged amino acids and/or amino acid analogs and B consists of 1 or 2 positively charged amino acids and/or amino acid analogs.
• the modified PNA molecules of formula I are used in the treatment or prevention of infections caused by Escherichia coli or vancomycin-resistant enterococci such as Enterococcus faecalis and Enterococcus faecium or infections caused by methicillin-resistant and methicillin-vancomycin- resistant Staphylococcus aureus.
• the peptide is linked to the PNA sequence via the amino (N-terminal) or carboxy (C- terminal) end.
• the peptide is linked to the PNA sequence via the carboxy end.
• the compounds of formula I may be prepared in the form of pharmaceutically acceptable salts, especially acid-addition salts, including salts of organic acids and mineral acids.
• salts include salts of organic acids such as formic acid, fumaric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid and the like.
• Suitable inorganic acid-addition salts include salts of hydrochloric, hydrobromic, sulphuric and phosphoric acids and the like.
• Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2 (1977) which are known to the skilled artisan.
• Also intended as pharmaceutically acceptable acid addition salts are the hydrates which the present compounds are able to form.
• the acid addition salts may be obtained as the direct products of compound synthesis.
• the free base may be dissolved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
• the compounds of this invention may form solvates with standard low molecular weight solvents using methods known to the skilled artisan.
• modified PNA molecules are used in the manufacture of medicaments for the treatment or prevention of infectious diseases or for disinfecting non-living objects.
• the invention concerns a composition for treating or preventing infectious diseases or disinfecting non-living objects.
• the invention concerns the treatment or prevention of infectious diseases or treatment of non-living objects.
• the present invention concerns a method of identifying specific advantageous antisense PNA sequences which may be used in the modified PNA molecule according to the invention.
• the present invention relates to other antisense oligonucleotides with the ability to bind to both DNA and RNA.
• Oligonucleotide analogues are oligomers having a sequence of nucleotide bases (nucleobases) and a subunit-to-subunit backbone that allows the oligomer to hybridize to a target sequence in an mRNA by Watson-Crick base pairing, to form an RNA/Oligomer duplex in the target sequence.
• the oligonucleotide analogue may have exact sequence complementarity to the target sequence or near complementarity, as long as the hybridized duplex structure formed has sufficient stability to block or inhibit translation of the mRNA containing target sequence.
• Oligonucleotide analogues of the present invention are selected from the group consisting of Locked Nucleoside Analogues (LNA) as described in International PCT Publication WO99/14226, oligonucleotides as described in International PCT Publication WO98/03533 or antisense oligomers, in particular morpholino analogues as described in International PCT Publication WO98/32467.
• LNA Locked Nucleoside Analogues
• L is a linker or a bond
• Peptide is any amino acid sequence
• Oligon designates an oligonucleotide or analogue thereof.
• FIGURE 1 shows the chemical structure of DNA and PNA oligomers.
• FIGURE 2 shows the principle in conjugation using SMCC
• FIGURE 3 shows the nucleotide sequence of the mrcA (ponA) gene encoding PBP1A.
• the sequence of the gene (accession number X02164) was obtained from the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al. 1985, Eur J Biochem 147:437-46 (41)). Two possible start codons have been identified (highlighted). Bases 1-2688 are shown (ending with stop codon).
• FIGURE 4 shows the nucleotide sequence of the mrdA gene encoding PBP2.
• the sequence (accession number AE000168, bases 4051-5952, numbered 1-2000) was obtained from the E. coli genome database at the NCBI (Genbank, National Centre for Biotechnology Information, USA). The start codon is highlighted.
• FIGURE 5 shows the chemical structures of the different succinimidyl based linking groups used in the conjugation of the Peptide and PNA
• Antisense PNA's can inhibit bacterial gene expression with gene and sequence specificity (Good and Nielsen 1998a,b (39,40) and WO 99/13893). The approach may prove practical as a tool for functional genomics and as a source for novel antimicrobial drugs. However, improvements on standard PNA are required to increase antisense potencies. The major limit to activity appears to be cellular entry. Bacteria effectively exclude the entry of large molecular weight foreign compounds, and previous results for in vitro and cellular assays seem to show that the cell barrier restricts antisense effects. Accordingly, the present invention concerns strategies to improve the activity of antisense potencies.
• the short cationic peptides lead to an improved PNA uptake over the bacterial cell wall. It is believed that the short peptides act by penetrating the cell wall, allowing the modified PNA molecule to cross the cell wall to get access to structures inside the cell, such as the genome, mRNA's, the ribosome, etc.
• an improved accessibility to the nucleic acid target or an improved binding of the PNA may also add to the overall effect observed.
• PNA molecules modified with short activity enhancing peptides enable specific and efficient inhibition of bacterial genes with nanomolar concentrations. Antisense potencies in this concentration are consistent with practical applications of the technology. It is believed that the present invention for the first time demonstrates that peptides with a certain pattern of cationic and lipophilic amino acids can be used as carriers to deliver agents and other compounds into micro-organisms, such as bacteria. Further, the present invention has made it possible to administer PNA in an efficient concentration, which is also acceptable to the patient.
• novel modified PNA molecules having the formula:
• L is a linker or a bond
• PNA is a peptide nucleic acid sequence
• Peptide is a cationic peptide or peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the formula:
• A consists of from 1 to 8 non-charged amino acids and/or amino acid analogs
• B consists of from 1 to 3 positively charged amino acids and/or amino acid analogs
• C consists of from 0 to 4 non-charged amino acids and/or amino acid analogs
• D consists of from 0 to 3 positively charged amino acids and/or amino acid analogs
• n 1-10; and the total number of amino acids and/or amino acid analogs is from 3 to 20.
• a preferred group of modified Peptide Nucleic Acids (PNA) molecule is the group wherein A consists of from 1 to 6 non-charged amino acids and/or amino acid analogs and B consists of 1 or 2 positively charged amino acids and/or amino acid analogs. In another preferred group A consists of from 1 to 4 non-charged amino acids and/or amino acid analogs and B consists of 1 or 2 positively charged amino acids and/or amino acid analogs.
• cationic amino acids and amino acid analogues and “positively charged amino acids and amino acid analogues” are to be understood any natural or non-natural occurring amino acid or amino acid analogue which have a positive charge at physiological pH.
• non-charged amino acids or amino acid analogs is to be understood any natural or non-natural occurring amino acids or amino acid analogs which have no charge at physiological pH.
• lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and ornithine (Orn).
• DAB diamino butyric acid
• Orn ornithine
• non-charged amino acids and amino acid analogs may be mentioned the natural occurring amino acids alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (lie, I), proline (Pro, P), phenylanaline (Phe, F), tryptophan (Trp, W), methionine (Met, M), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gin, Q), the non- natural occurring amino acids 2-aminobutyric acid, ⁇ -cyclohexylalanine, 4- chlorophenylalanine, norleucine and phenylglycine.
• the skilled person will be aware of further non-charged amino acids and amino acid analogs.
• the non-charged amino acids and amino acid analogs are selected from the natural occurring non-polar amino acids Ala, Val, Leu, lie, Phe, Trp and Met or the non-natural occurring non-polar amino acids ⁇ -cyclohexylalanine, 4- chlorophenylalanine and norleucine.
• the term "functionally similar moiety” is defined as to cover all peptide-like molecules, which functionally mimic the Peptide as defined above and thus impart to the PNA molecule the same advantageous properties as the peptides comprising natural and non-natural amino acids as defined above.
• Examples of preferred modified PNA molecules according to the invention are (Lys Phe Phe) 3 Lys-L-PNA and any subunits thereof comprising at least three amino acids.
• One preferred Peptide is (Lys Phe Phe) 3 (SEQ ID NO: 1).
• Others include (Lys Phe Phe) 2 Lys Phe (SEQ ID NO: 2), (Lys Phe Phe) 2 Lys (SEQ ID NO: 157), (Lys Phe Phe) 2 (SEQ ID NO: 3), Lys Phe Phe Lys Phe (SEQ ID NO: 4), Lys Phe Phe Lys (SEQ ID NO: 5) and Lys Phe Phe.
• FFRFFRFFR SEQ ID NO: 6
• LLKLLKLLK SEQ ID NO: 7
• LLRLLRLLR SEQ ID NO: 8
• LLKKLAKAL SEQ ID NO: 9
• KRRWPWWPWKK SEQ ID NO: 10
• KFKVKFVVKK SEQ ID NO: 11
• LLKLLLKLLLK SEQ ID NO: 12
• LLKKLAKALK SEQ ID NO: 13
• a third group of preferred Peptides is RRLFPWWWPFRRVC (SEQ ID NO: 14), GRRWPWWPWKWPLic (SEQ ID NO: 15), LVKKVATTLKKI FSKWKC (SEQ ID NO: 16), KKFKVKFVVKKC (SEQ ID NO: 17) and any subunit thereof comprising at least 3 amino acids whereof at least one amino acid is a positively charged amino acid.
• a fourth group of preferred Peptides is magainis (Zasloff, M., Proc. Natl. Acad. Sci. USA, 84, p. 5449-5453 (1987)), for instance the synthetic magainin derivative GIGKFLHAAKKFAKAFVAEIMNS-NH 2 (SEQ ID NO: 158) as well as ⁇ -amino-acid oligomers ( ⁇ -peptides) as described by Porter, E.A. et al, Nature, 404, p. 565, (2000).
• the number of amino acids in the peptide may be chosen between 3 and 20. It appears that at least 3 amino acids; whereof at least one is a positively charged amino acid is necessary to obtain the advantageous effect.
• the upper limit only seems to be limited by an upper limit of the overall size of the PNA molecule for the purpose of the practical use of said molecule.
• the total number of amino acids is 15 or less, more preferable 12 or less and most preferable 10 or less.
• the PNA molecule is connected to the Peptide moiety through a direct binding or through a linker.
• a variety of linking groups can be used to connect the PNA with the Peptide.
• linking groups may be advantageous in connection with specific combinations of PNA and Peptide.
• Preferred linking groups are ADO (8-amino-3,6-dioxaoctanoic acid), SMCC (succinimidyl 4-( ⁇ /-maleimidomethyl)cyclohexane-1-carboxylate) AHEX or AHA (6- aminohexanoic acid), 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, LCSMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido- caproate), MBS (succinimidyl m-maleimido-benzoylate), EMCS (succinimidyl N- ⁇ - maleimido-caproylate), SMPH (succinimidyl 6-( ⁇ -maleimido-propionamido) hexanoate, AMAS (succinimidyl N-( ⁇ -maleimido acetate),
• any of these groups may be used as a single linking group or together with more groups in creating a suitable linker. Further, the different linking groups may be combined in any order and number in order to obtain different functionalities in the linker arm.
• the linking group is a combination of the ⁇ .ALA linking group or the ADO linking group with any of the other above mentioned linking groups.
• preferred linkers are -achc- ⁇ .ala-, -ache-ado-, -Icsmcc- ⁇ .ala-, -mbs- ⁇ .ala-, - emcs- ⁇ .ala-, -lcsmcc-ado-, -mbs-ado-, -emcs-ado- or -smph-ado-.
• SMCC succinimidyl 4-( ⁇ /-maleimidomethyl)cyclohexane-1-carboxylate
• cysteine C
• amino acids such as glycine
• the Peptide is normally linked to the PNA sequence via the amino or carboxy end.
• the PNA sequence may also be linked to an internal part of the peptide or the PNA sequence is linked to a peptide via both the amino and the carboxy end.
• the modified PNA molecule according to the present invention comprises a PNA oligomer of a sequence, which is complementary to at least one target nucleotide sequence in a microorganism, such as a bacterium.
• the target may be a nucleotide sequence of any RNA, which is essential for the growth, and/or reproduction of the bacteria.
• the target may be a gene encoding a factor responsible for resistance to antibiotics.
• the functioning of the target nucleotide sequence is essential for the survival of the bacteria and the functioning of the target nucleic acid is blocked by the PNA sequence, in an antisense manner.
• the binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations, anti-parallel or parallel.
• the term complementary as applied to PNA does not in itself specify the orientation parallel or anti-parallel. It is significant that the most stable orientation of PNA/DNA and PNA/RNA is anti-parallel.
• PNA targeted to single strand RNA is complementary in an anti-parallel orientation.
• a bis-PNA consisting of two PNA oligomers covalently linked to each other is targeted to a homopurine sequence (consisting of only adenine and/or guanine nucleotides) in RNA (or DNA), with which it can form a PNA 2 -RNA (PNA 2 -DNA) triple helix.
• the PNA contains from 5 to 20 nucleobases, in particular from 7-15 nucleobases, and most particular from 8 to 12 nucleobases.
• Peptide Nucleic Acids are described in WO 92/20702 and WO 92/20703, the content of which is hereby incorporated by reference.
• the backbone is aminoethylglycine as shown in Figure 1.
• Target genes may be chosen based on the knowledge of bacterial physiology.
• a target gene may be found among those involved in one of the major process complexes: cell division, cell wall synthesis, protein synthesis (translation) and nucleic acid synthesis, fatty acid metabolism and gene regulation.
• a target gene may also be involved in antibiotic resistance.
• PBPs penicillin binding proteins
• beta-lactam antibiotic penicillin the targets of, e.g., the beta-lactam antibiotic penicillin. They are involved in the final stages of cross-linking of the murein sacculus.
• E. coli has 12 PBPs, the high molecular weight PBPs: PBP1a, PBP1b, PBP1c, PBP2 and PBP3, and seven low molecular weight PBPs, PBP 4-7, DacD, AmpC and AmpH.
• PBPs the high molecular weight PBPs
• PBP 4-7 the high molecular weight PBPs
• DacD the low molecular weight PBPs
• RNA synthesis Both DNA and RNA synthesis are target fields for antibiotics.
• a known target protein in DNA synthesis is gyrase. Gyrase acts in replication, transcription, repair and restriction.
• the enzyme consists of two subunits, both of which are candidate targets for PNA.
• Examples of potential targets primarily activated in dividing cells are rpoD, gyrA, gyrB, (transcription), mrcA (ponA), mrcB (ponB, pbpF), mrdA, ftsl (pbpB) (Cell wall biosynthesis), ftsQ, ftsA and tifsZ (cell division).
• Examples of potential targets also activated in non-dividing cells are infA, infB, infC, tufA/tufB, tsf, fusA, prfA, prfB, and prfC, (Translation).
• antibiotic resistance-genes Other potential target genes are antibiotic resistance-genes. The skilled person would readily know from which genes to choose. Two examples are genes coding for beta-lactamases inactivating beta-lactam antibiotics, and genes encoding chloramphenicol acetyl transferase.
• PNA's against such resistance genes could be used against resistant bacteria.
• a further potential target gene is the acpP gene encoding the acyl carrier protein of E. Coli
• ACP acyl carrier protein
• ACP is a small and highly soluble protein, which plays a central role in type I fatty acid synthase systems.
• Intermediates of long chain fatty acids are covalently bound to ACP by a thioester bond between the carboxyl group of the fatty acid and the thiol group of the phosphopanthetheine prosthetic group.
• ACP is one of the most abundant proteins in E. coli, constituting 0.25% of the total soluble protein (ca 6 x 10 4 molecules per cell).
• the cellular concentration of ACP is regulated, and overproduction of ACP from an inducible plasmid is lethal to E. coli cells.
• Infectious diseases are caused by micro-organisms belonging to a very wide range of bacteria, viruses, protozoa, worms and arthropods and from a theoretical point of view PNA can be modified and used against all kinds of RNA in such microorganisms, sensitive or resistant to antibiotics.
• micro-organisms which may be treated in accordance with the present invention are Gram-positive organisms such as Streptococcus, Staphylococcus, Peptococcus, Bacillus, Listeria, Clostridium, Propionebacteria, Gram-negative bacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella, Salmonella, Shigella, Proteus, Pseudomonas, Vibrio, Legionella, Haemophilus, Bordetella, Brucella, Campylobacter, Neisseria, Branhamella, and organisms which stain poorly or not at all with Gram's stain such as Mycobacteria, Treponema, Leptospira, Borrelia, Mycoplasma, Clamydia, Rickettsia and Coxiella,
• VRE vancomycin resistant enterococci
• Coagulase negative staphylococci such as S. epidermidis are an important cause of infections associated with prosthetic devices and catheters (13). Although they display lower virulence than S .aureus, they have intrinsic low-level resistance to many antibiotics including beta-lactams and glycopeptides. In addition many of these bacteria produce slime (biofilm) making the treatment of prosthetic associated infections difficult and often requires removal of the infected prosthesis or catheter (24).
• Streptococcus pneumoniae regarded as fully sensitive to penicillin for many years, has now acquired the genes for resistance from oral streptococci. The prevalence of these resistant strains is increasing rapidly worldwide and this will limit the therapeutic options in serious pneumococcal infections, including meningitis and pneumonia (10). Streptococcus pneumoniae is the leading cause of infectious morbidity and mortality worldwide. In USA the pneumococcus is responsible for an estimated 50.000 cases of bacteremia, 3000 cases of meningitis, 7 million cases of otitis media, and several hundred thousands cases of pneumonia. The overall yearly incidence of pneumococcal bacteremia is estimated to be 15 to 35 cases per 100.000. Current immunization of small children and old people have not addressed the high incidence of pneumococcal infection ( 27, 28 ). Multi-drug resistant strains were isolated in the late 1970's and are now encountered worldwide (10)
• Cystitis, pneumonia, septicaemi and postoperative sepsis are the commonest types of infections. Most of the infections in patients being treated on an intensive care unit (ICU) results from the patients own endogenous flora and in addition up to 50% of ICU patients will also acquire nosocomial infection, which are associated with a relatively high degree of morbidity and mortality (19, 11 , 12). Microorganisms associated with these infections include Enterobacteriaceae 34%, S. aureus 30%, P. aeruginosa 29%, CNS 19% and fungi 17%.
• ICU intensive care unit
• the cell envelope of P. aeruginosa with the low permeability differs from that of E. coli. 46% of P. aeruginosa isolates from Europe are resistant to one or more antibiotics and the ability of this bacteria to produce slime (biofilm) and rapid development of resistance during treatment often leads to therapy failure. Multidrug resistant P. aeruginosa has also become endemic within some specialised ICU's such as those treating burns patients and cystic fibrosis patients (15, 16)
• RESULTS The ability of the compounds of the present invention to inhibit bacterial growth may be measured in many ways, which should be clear to the skilled person.
• the bacterial growth is measured by the use of a microdilution broth method according to NCCLS guidelines.
• the present invention is not limited to this way of detecting inhibition of bacterial growth.
• Bacterial strain E.coli K12 MG1655
• a logphase culture of E.coli is diluted with fresh preheated medium and adjusted to defined OD (here: Optical Density at 600 nm) in order to give a final concentration of 5x10 5 and 5x10 4 bacteria/ml medium in each well, containing 200 ⁇ l of bacterial culture.
• PNA is added to the bacterial culture in the wells in order to give final concentrations ranging from 300 nM to 1000 nM.
• Trays are incubated at 37°C by shaking in a robot analyzer, PowerWave * , software KC 4, Kebo.Lab, Copenhagen, for 16 h and optical densities are measured at 600 nM during the incubation time in order to record growth curves.
• Wells containing bacterial culture without PNA are used as controls to ensure correct inoculum size and bacterial growth during the incubation. Cultures are tested in order to detect contamination.
• the individual peptide-L-PNA constructs have MW between approx. 4200 and 5000 depending on the composition. Therefore all tests were performed on a molar basis rather than on a weight/volume basis. However, assuming an average MW of the construct of 4500 a concentration of 500 nM equals 2.25 microgram/ml.
• the bacterial growth in the wells is described by the lag phase i.e. the period until (before) growth starts, the log phase i.e. the period with maximal growth rate, the steady-state phase followed by the death phase. These parameters are used when evaluating the inhibitory (Minimal Inhibitory Concentration, abbr. MIC) and bactericidal (Minimal Bactericidal Concentration, abbr. MBC) effect of the PNA on the bacterial growth, by comparing growth curves with and without PNA.
• inhibitory Minimal Inhibitory Concentration, abbr. MIC
• bactericidal Minimal Bactericidal Concentration, abbr. MBC
• OD (16h) OD (Oh) or no visible growth according to NCCLS Guidelines
• modified PNA molecules are tested in the sensitive 10% medium assay. Positive results are then run in the 100% medium assay in order to verify the inhibitory effect in a more "real" environment (cf. the American guidelines (NCCLS)).
• NCCLS American guidelines
• the modified PNA molecules can be used to identify preferred targets for the PNA. Based upon the known or partly known genome of the target micro-organisms, e.g. from genome sequencing or cDNA libraries, different PNA sequences can be constructed and linked to an effective anti-infective enhancing Peptide and thereafter tested for its anti-infective activity. It may be advantageous to select PNA sequences shared by as many microorganisms as possible or shared by a distinct subset of micro-organisms, such as for example Gram-negative or Gram-positive bacteria, or shared by selected distinct micro-organisms or specific for a single micro-organism.
• the invention provides a composition for use in inhibiting growth or reproduction of infectious micro-organisms comprising a modified PNA molecule according to the present invention.
• the inhibition of the growth of micro-organisms is obtained through treatment with either the modified PNA molecule alone or in combination with antibiotics or other anti- infective agents.
• the composition comprises two or more different modified PNA molecules.
• a second modified PNA molecule can be used to target the same bacteria as the first modified PNA molecule or in order to target different bacteria.
• specific combinations of target bacteria may be selected to the treatment.
• the target can be one or more genes, which confer resistance to one or more antibiotics to one or more bacteria.
• the composition or the treatment further comprises the use of said antibiotic(s).
• the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of the general formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.
• compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practise of Pharmacy, 19 th Ed., 1995.
• the compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.
• compositions include a compound of formula I or a pharmaceutically acceptable acid addition salt thereof, associated with a pharmaceutically acceptable excipient which may be a carrier or a diluent or be diluted by a carrier, or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container.
• a pharmaceutically acceptable excipient which may be a carrier or a diluent or be diluted by a carrier, or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container.
• the active compound will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a ampoule, capsule, sachet, paper, or other container.
• the carrier When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material which acts as a vehicle, excipient, or medium for the active compound.
• the active compound can be adsorbed on a granular solid container for example in a sachet.
• suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatine, lactose, terra alba, sucrose, glucose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone.
• the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
• the formulations may also include wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, thickeners or flavouring agents.
• the formulations of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
• compositions can be sterilized and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring substances and the like, which do not deleteriously react with the active compounds.
• the route of administration may be any route, which effectively transports the active compound to the appropriate or desired site of action, such as oral, nasal, rectal, pulmonary, transdermal or parenteral e.g. depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment, the parenteral or the oral route being preferred.
• the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge.
• a liquid carrier is used, the preparation may be in the form of a suspension or solution in water or a non-aqueous media, a syrup, emulsion or soft gelatin capsules. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be added.
• the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application.
• a liquid carrier in particular an aqueous carrier
• the carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
• solubilizing agents e.g. propylene glycol
• surfactants e.g. propylene glycol
• absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin
• preservatives such as parabenes.
• injectable solutions or suspensions preferably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.
• Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or binder or the like are particularly suitable for oral application.
• Preferable carriers for tablets, dragees, or capsules include lactose, corn starch, and/or potato starch.
• a syrup or elixir can be used in cases where a sweetened vehicle can be employed.
• the amount of active modified PNA molecules used is determined in accordance with the specific active drug, organism to be treated and carrier of the organism.
• Such mammals include also animals, both domestic animals, e.g. household pets, and non-domestic animals such as wildlife.
• dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.01 mg to about 500 mg, preferably from about 0.01 mg to about 100 mg of the compounds of formula I admixed with a pharmaceutically acceptable earner or diluent.
• the present invention relates to the use of one or more compounds of the general formula I or pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment and/or prevention of infectious diseases.
• the present invention concerns a method of treating or preventing infectious diseases, which treatment comprises administering to a patient in need of treatment or for prophylactic purposes an effective amount of modified PNA according to the invention.
• a treatment may be in the form of administering a composition in accordance with the present • invention.
• the treatment may be a combination of traditional antibiotic treatment and treatment with one or more modified PNA molecules targeting genes responsible for resistance to antibiotics.
• the present invention concerns the use of the modified PNA molecules in disinfecting objects other than living beings, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools and the like.
• linking groups as starting materials are indicated with capital letters whereas the linking groups in the finished peptide-PNA conjugate are indicated with small letters.
• linking groups containing a succinimidyl group are shown in Figure 5. All the linking groups are commercial available.
• the composition of mixtures of solvents is indicates on a volume basis, i.e. 30/2/10 (v/v/v).
• Preparative HPLC is performed on a DELTA PAK [Waters ](C18,15 ⁇ m, 300 A, 300x7.8 mm, 3 ml/min)
• H-KFFKFFKFFK-ado- ⁇ c AAA CAT AGT-NH ? The peptide-PNA-chimera H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NH 2 (SEQ ID NO: 18) was synthesized on 50 mg MBHA resin (loading 100 ⁇ mol/g) (novabiochem) in a 5 ml glass reactor with a D-2 glassfilter. Deprotection was done with 2x600 ⁇ L TFA/m-cresol 95/5 followed by washing with DCM, DMF, 5% DIEA in DCM and DMF.
• the coupling mixture was 200 ⁇ l 0.26 M solution of monomer (Boc- PNA-T-monomer, Boc-PNA-A-monomer, Boc-PNA-G-monomer, Boc-PNA-C- monomer, Boc-AEEA-OH (ado) (PE Biosystems Inc.)) in NMP mixed with 200 ⁇ l 0.5 M DIEA in pyridine and activated for 1 min with 200 ⁇ l 0.202 M HATU (PE- biosystems) in NMP.
• monomer Boc- PNA-T-monomer, Boc-PNA-A-monomer, Boc-PNA-G-monomer, Boc-PNA-C- monomer, Boc-AEEA-OH (ado) (PE Biosystems Inc.)
• NMP NMP mixed with 200 ⁇ l 0.5 M DIEA in pyridine and activated for 1 min with 200 ⁇ l 0.202 M HATU (PE- biosystems) in NMP.
• the coupling mixture for the peptide part was 200 ⁇ l 0.52 M NMP solution of amino acid (Boc-Phe-OH and Boc-Lys(2-CI-Z)-OH (novabiochem)) mixed with 200 ⁇ l 1 M DIEA in NMP and activated for 1 min with 200 ⁇ l 0.45 M HBTU in NMP.
• the resin was washed with DMF, DCM and capped with 2 x 500 ⁇ l NMP/pyridine/acetic anhydride 60/35/5. Washing with DCM, DMF and DCM terminated the synthesis cycle.
• the oligomer was deprotected and cleaved from the resin using "low-high" TFMSA.
• the resin was rotated for 1 h with 2 ml of TFA/dimethylsulfid/ m-cresol/TFMSA 10/6/2/0.5. The solution was removed and the resin was washed with 1 ml of TFA and added 1.5 ml of TFMSA/TFA/m- cresol 2/8/1. The mixture was rotated for 1.5 h and the filtrated was precipitated in 8 ml diethylether. The precipitate was washed with 8 ml of diethylether. The crude oligomer was dissolved in water and purified by HPLC.
• Preparative HPLC was performed on a DELTA PAK [Waters ](C18,15 ⁇ m, 300 A, 300x7.8 mm, 3 ml/min)
• PNA-oligomer ado- ⁇ c AAA CAT AGT-NH 2 (SEQ ID NO: 19) (purified by HPLC) (2 mg, 0.589 ⁇ mol, Mw 3396.8) was dissolved and stirred for 15 min in NMP:DMSO 8:2 (2 ml).
• Succinimidyl 4-( ⁇ /-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (PIERCE)(1.1 mg, 3.24 ⁇ mol, 5.5 eq.) dissolved in NMP (50 ⁇ l) and DIEA (34.7 ⁇ l, 198.7 ⁇ mol) was added to the solution: The reaction mixture was stirred for further 2.5 h.
• the product was precipitated in diethylether (10 mL). The precipitate was washed with ether:NMP; 10:1(3x10mL) and ether (3x10mL). Mw calculated: 3615.8 g/mol; found on MALDI: 3613.5 g/mol. The product was used without further purification.
• Preparative HPLC was performed on a DELTA PAK [Waters ](C18,15 ⁇ m, 300 A, 300x7.8 mm, 3 ml/min)
• Mw calculated: 5133.0 g/mol; found on MALDI: 5133 g/mol.
• PNA oligomer ado-JTJTJJT-ado-ado-ado-ado- ⁇ ccc ⁇ c ⁇ c-Lys-NH 2 (SEQ ID NO: 23) instead of ado- ⁇ c AAA CAT AGT-NH 2 (SEQ ID NO: 19).
• This PNA is a triplex forming bis- PNA in which C (cytosine) in the "Hoogsteen strand" is exchanged with the J nucleobases (a substitute for protonated C). This substitution assures efficient triplex formation at physiological pH (Egholm, M.; Dueholm, K. L; Buchardt, O.; Coull, J.; Nielsen, P. E.; Nucleic Acids Research 1995, 23,217-222 (42)).
• the peptide-PNA-chimeras in Table III were prepared as described in Example 1 using the linking groups as defined above.
• the bacterial growth assay is designed to identify modified PNA molecules that inhibit or completely abolish bacterial growth. Growth inhibition results from antisense binding of PNA to mRNA of the targeted gene. The compound tested is present during the whole assay.
• the experimental bacterial strain for the protocol is Escherichia coli K12 MG1655 (E. coli Genentic Stock Center, Yale University, New Haven).
• the medium for growth is 10% sterile LB (Lurea Bertani) medium.
• E. coli test cells are pre-cultured in LB medium at 37 °C over night (over night culture).
• the screen is performed in 96-well microtiter plates at 37 °C under constant shaking.
• PNA's are dissolved in H 2 0 as a 40x concentrated stock solution.
• the test culture is diluted stepwise in the range 10 s to 10 1 with 10% LB medium. 195 ⁇ l of diluted cultures plus 5 ⁇ l of a 40x concentrated PNA stock solution are added to each test well.
• 96-well microtiter plates are incubated in a microplate scanning spectrophotometer at 37 °C under constant shaking. OD 6 oo measurements are performed automatically every 3.19 minutes and recorded simultaneously.
• PBPs Penicillin binding proteins
• PBPs act in biosynthesis of murein (peptidoglycan), which is part of the envelope of Gram-positive and Gram-negative bacteria.
• peptidoglycan peptidoglycan
• PBP's By binding of penicillin, which acts as substrate analogue, PBP's are inhibited, and subsequently, hydrolytic enzymes are activated by the accumulation of peptidoglycan intermediates, thus hydrolysing the peptidoglycan layer and causing lysis.
• E.coli has 7-9 PBPs, the high molecular weight PBPs, PBP1A and PBP1 B, PBP2 and PBP3, and the low molecular weight PBPs, PBP 4-9.
• the high molecular weight PBPs are essential for growth, whereas the low molecular weight PBPs are not essential.
• PNA26 has been designed according to the sequence of the mrcA (ponA) gene of E. coli, encoding PBP1A.
• the sequence of the mrcA gene (accession number X02164) was obtained from the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al. 1985, Eur J Biochem 147:437-46 (41)).
• the sequence of the mrcA gene is shown in Figure 3.
• the target region of PNA26 is the following:
• PNA26 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide.
• Dilutions of the test culture corresponding to 10 5 , 10 4 , 10 3 , 10 2 and 10 1 cells/ml containing PNA26 at a final concentration of 1.5, 2.0, 2.5, 3.0 and 3.5 ⁇ M are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with 10 4 -10 1 cells/ml and a PNA concentration of at least 2.5 ⁇ M (Table 1).
• PNA 14 has been designed according to the sequence of the mrdA gene encoding PBP2.
• the sequence (accession number AE000168, bases 4051-5952) was obtained from the E. coli genome database at the NCBI (Genbank, National Centre for Biotechnology Information, USA).
• the sequence of the mrdA gene is shown in Figure 4
• the target region of PNA14 is the following:
• PNA14 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide.
• Dilutions of the test culture corresponding to 10 5 , 10 4 , 10 3 , 10 2 and 10 1 cells/ml containing PNA14 at a final concentration of 1.3, 1.4 and 1.5 ⁇ M are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with 10 4 -10 1 cells/ml and a PNA concentration of at least 1.4 ⁇ M (Table 2).
• Peptides are truncated versions of the KFF-motif.
• the basic peptide sequence is KFFKFFKFFK (SEQ ID NO: 148) (PNA 1).
• PNA 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 all contain peptides which are truncated from the C-terminal end.
• PNA 84, 85, 86, 87, 88, 89, 90, 91 and 92 all contain peptides which are truncated from the N-terminal end.
• the PNA against the LacZ-gene has been synthesized with and without an - NH 2 terminal lysine.
• the assay was performed as follows:
• Dilutions of the test culture E. coli K12 corresponding to, 5x10 5 and 5x10 4 , cells/ml containing truncated versions of the KFF-motif of the PNA's against the LacZ gene at a final concentration of 100, 300, 750 and 1500 nM are incubated in M9 minimal broth with lactose as the sole carbon source (minimal media 9, Bie & Bemtsen Cph) at 37°C for 16 hours with constant shaking.
• the PNA130 and 218-226 against the infA-gene have been synthesized with peptides as truncated versions of the KFF-motif.
• Dilutions of the test culture E. coli K12 corresponding to, 2x10 4 and 4x10 4 , cells/ml containing truncated versions of the KFF-motif of the PNA's against the infA-gene at a final concentration of 200, 400, 600 800 and 1000 nM are incubated in 10% Mueller-Hinton broth at 37°C for 16 hours with constant shaking.
• the assay was performed as follows:
• Dilutions of the test culture E. coli K12 corresponding to, 2x10 4 and 4x10 4 , cells/ml containing truncated versions of the KFF-motif of the PNA's against ⁇ -sarcine loop of ribosomal RNA at a final concentration of 200, 400, 600, 800 and 1000 nM are incubated in 10% Mueller-Hinton broth at 37°C for 16 hours with constant shaking.
• Dilutions of the test culture E. coli K12 corresponding to, 700 and 350 cells/ml containing variations of amphipathie 10, 11 and 12-mer structures with smcc-linker of the PNA's against the FtsZ-gene at a final concentration of 200, 300, 400, 500, 600, 800 and 1000 nM are incubated in 100% Mueller-Hinton broth at 37°C for 16 hours with constant shaking.
• the sequence of the nucleobases is the same as the sequence in PNA 130, but the linking groups and the peptides varies.
• the smcc-ado linker seems to be the superior linker showing total inhibition of growth in cultures with 1.6x10 3 -8x10 2 cells/ml and a PNA concentration of at least 600nM.
• the peptide no. 2339 with the sequence: H-KFFKFFKFF-OH (SEQ ID NO: 1) was added to E. coli K12 in 10% and 100% medium (Mueller-Hinton broth). Growth assay of the peptide no. 2339 The assay was performed as follows:
• the assay was performed as follows:
• (+) Significantly extended lagphase, (more than five times)
• ((+)) Lagphase extended less than five times, but still with growth curve effect
• (+) Significantly extended lagphase, (more than five times)
• ((+)) Lagphase extended less than five times, but still with growth curve effect
• PNA 249 is equal with PNA 109, without the peptide but still with the ado-linker.
• the Peptide of PNA 250 has the sequence: H-CG-KLAKALKKLL-NH 2 (SEQ ID NO: 156).
• the peptide is also used for PNA 174.
• PNA Bacterial growth inhibition with PNA against the gene encoding IF-1 of E. coli. E. coli K12 in 10% Mueller-Hinton broth. Peptides are versions of the KFF-motif placed C- or N-terminal to the PNA.
• the orientation of the Peptide is not so important. However, for specific combinations of PNA and Peptide, one of the orientations may be preferred.
• E. faecium genome is, alongside with 250 other genomes, commercially available from Integrated Genomics, Chicago.
• PNA conjugates to inhibit bacterial growth is measured by the use of a microdilution broth method using 100% Mueller-Hinton broth, according to NCCLS Guidelines.
• a logphase culture of E. faecium is diluted with fresh prewarmed medium and adjusted to defined OD (here: Optical Density at 600 nm) in order to give a final concentration of 1x10 4 bacteria/ml medium in each well, containing 195 ⁇ l of bacterial culture.
• PNA is added to the bacterial culture in the wells in order to give final concentrations ranging from 450 nM to 1500 nM.
• Trays e.g.
• Costar#3474 are incubated at 35°C by shaking in a robot analyzer (96 well microtiter format), PowerWave x , software KC 4, Kebo.Lab, Copenhagen, for 16 h and optical densities are measured at 600 nm at short intervals during the incubation time in order to record growth curves. All cultures are tested in order to detect contaminations.
• ATCC 51550 Multidrugresistant (ampicillin, ciprofloxacin, gentamycin, rifampin, teicoplanin, vancomycin)
• PNA conjugate from Example 20 Bacterial strains: 8803, 51550, 700221 PNA concentration in wells: 400, 800 and 1600 nM
• MIC ' s The Minimal Inhibitory Concentrations (MIC ' s) of the PNA conjugate were as follows:
• a peptide-PNA-chimera was prepared in the same way as described in Example 1 : H 2 N-KKFKVKFWKKC-smcc-Ado-ACTTTGTCGCCC-NH 2 (SEQ ID NO: 160) .
• Staphylococcus aureus NCTC 8325 This strain was obtained from Prof. J. landolo, University of Oklahoma Health Sciences Center, Department of Microbiology and Immunology. S.aureus NCTC 8325 is being sequenced in the S. aureus Genome Sequencing Project at the University of Oklahoma's Advanced Center for Genome Technology (OU-ACGT).
• the genome is not completely sequenced.
• the genome size is 2.80 Mb, of which a total of 2,581 ,379 bp has been sequenced.
• Annotated gene sequences are available from Genbank for a number of putative targets.
• the antibacterial PNA conjugate prepared in Example 22 was used for the following experiments:
• PNA PNA to inhibit bacterial growth is measured by the use of a microdilution broth method using 100% Mueller-Hinton broth, according to NCCLS Guidelines.
• a logphase culture of S aureus is diluted with fresh pre warmed medium and adjusted to defined OD (here: Optical Density at 600 nm) in order to give a final concentration of 1x10 4 bacteria/ml medium in each well, containing 195 ⁇ l of bacterial culture.
• PNA is added to the bacterial culture in the wells in order to give final concentrations ranging from 450 nM to 1500 nM. Trays (e.g.
• Costar#3474 are incubated at 35°C by shaking in a robot analyzer (96 well microtiter format), PowerWave x , software KC 4, Kebo.Lab, Copenhagen, for 16 h and optical densities are measured at 600 nm at short intervals during the incubation time in order to record growth curves. All cultures are tested in order to detect contaminations.
• ATCC 700698R highly vancomycin resistant subclone of 11 ATCC 700698 Experimental setup :
• MCC's inhibited bacteria
• MIC Minimal Inhibitory Concentrations
• a compound of the invention was tested for antibacterial effect in vivo according to the test described by N. Frimodt-M ⁇ ller.
• Levy SB Balancing the drug resistance equation. Trends Microbial 1996; 2: 341-2 2.
• Levy SB The antibiotic paradox, how miracle drugs are destroying the miracle. New York: Plenum, 1992

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• 2000
  • 2000-10-13 WO PCT/DK2000/000580 patent/WO2001027261A2/en not_active Application Discontinuation
  • 2000-10-13 IL IL14909500A patent/IL149095A0/xx unknown
  • 2000-10-13 EP EP00967618A patent/EP1220902A2/de not_active Withdrawn
  • 2000-10-13 HU HU0203465A patent/HUP0203465A2/hu unknown
  • 2000-10-13 CA CA002388991A patent/CA2388991A1/en not_active Abandoned
  • 2000-10-13 AU AU77730/00A patent/AU7773000A/en not_active Abandoned
  • 2000-10-13 JP JP2001530466A patent/JP2003511466A/ja active Pending
• 2002
  • 2002-04-11 NO NO20021711A patent/NO20021711L/no not_active Application Discontinuation

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