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WO2002053574A2 - Molecules pna modifiees - Google Patents

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Publication number
WO2002053574A2
WO2002053574A2 PCT/DK2002/000005 DK0200005W WO02053574A2 WO 2002053574 A2 WO2002053574 A2 WO 2002053574A2 DK 0200005 W DK0200005 W DK 0200005W WO 02053574 A2 WO02053574 A2 WO 02053574A2
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pna
mmol
solution
vacuo
added
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PCT/DK2002/000005
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WO2002053574A3 (fr
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Peter Eigil Nielsen
Muthiah Manoharan
Ask Püschl
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Pantheco A/S
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Priority to AU2002216946A priority Critical patent/AU2002216946A1/en
Publication of WO2002053574A2 publication Critical patent/WO2002053574A2/fr
Publication of WO2002053574A3 publication Critical patent/WO2002053574A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention concerns novel drugs for use in combating infectious microorganisms, in particular bacteria. More particular the invention concerns peptide nucleic acid (PNA) se- quences, which are optionally modified in order to obtain novel PNA molecules with anti- infective properties.
  • PNA peptide nucleic acid
  • 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. One of these is duplex formation between an oligonucleotide and a single stranded nucleic acid. Another is tri- plex formation between an oligonucleotide and double stranded DNA to form a triplex struc- ture.
  • PNA Peptide nucleic acids
  • oligonucleotides are compounds that in certain respects are similar to oligonucleotides and their analogs and thus may mimic DNA and RNA.
  • the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 (2)) (Fig. 1 ).
  • Each subunit, or monomer has a naturally occurring or non-naturally occurring nucleobase attached to this backbone.
  • 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 (3)).
  • the Pseudo-peptide backbone provides superior hybridization properties (Egholm et al. 1993), resistance to enzymatic degradation (Demidov et al. 1994 (4)) and access to a variety of chemical modifications (Nielsen and Haaima 1997 (5)).
  • 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. In addition to increased affinity, 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
  • PNA polyamide backbone having appropriate nucleobases or other side chain groups attached thereto
  • 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 (6), Knudsen et al. 1996 (7), Good and Nielsen 1998 (12)), 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 (8)), encapsulation (Meyer et al. 1998 (9)) and carrier strategies (Nyce and Metzger 1997 (10), Pooga et al, 1998 (11)).
  • 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 rec- ognising 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 (12, 13), (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, ter- penes, 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 or amidino sidechain moiety and wherein at least 6 contiguous subunits contain guanidino and/or amidino sidechains.
  • a preferred transporter polymer is a polypeptide containing 9 ar- ginine.
  • WO 98/03542 discloses Peptide nucleic acids having enhanced binding affinity including PNAs with amino acid side chain modifications.
  • E. Coli AS19 were 10 times as sensitive towards the PNA as wildtype E. Coli K-12, indicating that uptake was responsible for the modest (but significant) antibacterial effect. Similar a triplex forming bis-PNA targeting the almost single stranded peptidyl transferase center showed almost the same effect. Duplex forming PNAs (targeting the peptidyl transferase center or the mRNA binding region of 16S rRNA) did not show any effect below 0.5 ⁇ M in vitro, and below 20 ⁇ M concentration in vivo.
  • the alpha-sarcin loop contains the longest universally conserved sequence (12 nt) of all rRNA and is therefore not a good target for an antibacterial PNA (Meyer et al. 1996 (17)).
  • the peptidyl transferase center is a possible target though.
  • the ⁇ -lactamase gene codes for the ⁇ -lactamase enzyme which cleaves the antibiotic pennicilin (which contains a four-membered lactam ring). This gene is not part of the E. Coli chromosome. Instead it is a plasmid (a small piece of circular dsDNA) taken up by the E. Coli from the outside.
  • One way of killing resistant bacteria could be to inhibit protein synthesis of the ⁇ -lactamase enzyme with an antisense PNA.
  • the completed nucleotide sequence of the ⁇ -lactamase gene was published in 1978 ( Figure 6) (Sutcliffe, 1978 (19))
  • This gene codes for a protein of 286 amino acids. By reading the coding strand, the following appears: The E. Coli RNA polymerase binds at the promotor sequence containing the so-called Pribnow box (indicated) and transcribes the gene to a mRNA copy. The ribosome starts the protein synthesis from the mRNA at the start codon ATG (indicated). This area is the best place to target the duplex forming antisense PNA. The 12-mer PNA sequence used in this study is underlined.
  • PNA 1438 was a 15 mer with 3 more residues in the C-terminal part
  • PNA 1439 had 3 more residues in the N terminal part of the PNA used in this study.
  • the first 23 amino acids are believed to be a secretion signal since this hydrophobic peptide is not part of the mature enzyme.
  • no triplex forming bis PNAs were targeted against any of the three 7 nt homo purine targets available in the mRNA (indicated in the coding strand).
  • the present invention relates to modifications of the PNAs improving the uptake of the PNAs. 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. Surprisingly, it has been found out that by incorporating a peptide, an enhanced anti-infective effect can be observed.
  • modified PNA molecules seems to be a pattern comprising in particular positively charged and lipophilic amino acids or amino acid analogues.
  • An anti-infective effect is found with different orientation of the peptide in relation to the PNA-sequence.
  • B is a naturally-occurring nucleobase or a non-naturally-occurring nucleobase
  • (Pr) is hydrogen or a protection group; and R is C- ⁇ - 6 -alkyl, 3-guanidinopropyl, carboxymethyl, aminocarboxymethyl, mercaptomethyl,
  • Q - L - PNA (I) wherein L is a linker or a bond; Q is a peptide and PNA is a peptide nucleic acid oligomer with from 6 to 25 monomers selected from the group consisting of aeg-PNA monomer and a monomer of formula (II).
  • the protection group is selected from, Boc, (tert-butyloxycarbonyl), Cbz (benzyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Mmt (monomethoxytrityl) or another group known by those skilled in the art.
  • the modified PNA molecules are used in the manufacture of medicaments for the treatment or prevention of a disease selected from bacterial and viral infections, cardiac or vascular diseases, metabolic diseases or immunological disorders 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 ac- cording to the invention.
  • FIGURE 1 shows the chemical structure of DNA and PNA oligomers.
  • the PNA oligomer has a backbone constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
  • the repeating unit is designated "aeg”.
  • Each monomer unit designated “aeg-PNA monomer” is also shown.
  • 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, EurJ Biochem 147:437-46 (14)). 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 Informa- tion, 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
  • FIGURE 6 shows the sequence of the ⁇ -Lactamase gene.
  • the top strand represents the coding strand (5'-3') and the bottom strand represents the template strand (3'-5').
  • FIGURE 7 Bar graph showing the anti ⁇ -Lactamase activity of the PNA's (Table 1 ) in £. Coli K12 at 1 ⁇ M conc.
  • FIGURE 8 Bar graph showing the anti ⁇ -Lactamase activity of the PNA's (Table 1 ) in £. Coli K12 at 0.5 ⁇ M conc.
  • FIGURE 9 Bar graph showing the anti ⁇ -Lactamase activity of the PNA's (Table 1) in E. Coli AS19 at 1 ⁇ M cone.
  • FIGURE 10 Bar graph showing the anti ⁇ -Lactamase activity of the PNA's (Table 1 ) in E. Coli AS19 at 0.5 ⁇ M conc.
  • FIGURE 11 Graph showing the antisense effect of normal aminoethylglycine PNA (1833) compared to the corresponding PNA (1883) in which three positions (T Lys ) have been replaced by a lysine-T PNA monomer (R being 4-aminobutyl).
  • the PNAs were targeted to the translation initiation region of the E. coli beta-galactosidase gene (lacZ). Cultures of E. coli were incubated with the indicated concentrations of PNA, and the beta-galactosidase activity was measured after overnight growth.
  • Antisense PNA's can inhibit bacterial gene expression with gene and sequence specificity (Good and Nielsen 1998a,b (12, 13) 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 molecu- lar 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 tech- nology. 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.
  • Ci-e-alkyl represent a branched or straight alkyl group having from one to six carbon atoms.
  • Typical Ci-e-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso- hexyl and the like.
  • 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.
  • positively charged amino acids and amino acid analogs may be mentioned lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and omithine (Orn).
  • lysine Lysine
  • Arg, R arginine
  • DAB diamino butyric acid
  • Orn omithine
  • 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.
  • 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 .
  • Others are (Lys Phe Phe) 2 Lys Phe, (Lys Phe Phe) 2 Lys, (Lys Phe Phe) 2 , Lys Phe Phe Lys Phe, Lys Phe Phe Lys and Lys Phe Phe as disclosed in PCT Publication WO 01/27261.
  • 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 prefer- able 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 are described in WO 96/11205, WO 01/27261 and WO98/52614, the content of which are hereby incorporated by reference. Some 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.
  • SMCC succinimidyl 4-( ⁇ /-maleimidomethyl)cyclohexane-1 -carboxylate
  • C cysteine
  • thiol containing moiety amino ac- ids, such as glycine, may be a part of the linker.
  • the Peptide is normally linked to the PNA sequence via the amino or carboxy end. However, the PNA sequence may also be linked to an internal part of the peptide.
  • 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 micro- organism, 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 oli- gomers 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 nucleo- bases, 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.
  • Target genes may be chosen based on the knowledge about bacterial physiology.
  • a target gene may be found among those involved in one of the four major process complexes: cell division, cell wall synthesis, protein synthesis (translation) and nucleic acid synthesis.
  • 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, PBP1 b, PBP1c, PBP2 and PBP3, and seven low molecular weight PBPs, PBP 4-7, DacD, AmpC and AmpH.
  • PBP1a the high molecular weight PBPs
  • PBP1b the high molecular weight PBPs
  • PBP1c the high molecular weight
  • PBP2 and PBP3 seven low molecular weight PBPs
  • PBP 4-7 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 ftsZ (cell division).
  • Examples of potential targets also activated in non-dividing cells are infA, infB, infC, MA/tufB, tsf, fusA, prfA, prfB, and priC, (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.
  • 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 micro-organisms, 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, Bacil- lus, Listeha, Clostndium, Propionebacteria, Gram-negative bacteria such as Bacteroides,
  • the ability of PNA's 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 x , 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 dur- ing 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 concen- tration of 500 nM equals 2.25 microgram/ml.
  • lag phase i.e. the period until growth starts
  • log phase i.e. the period with maximal growth rate
  • steady-state phase followed by death phase.
  • 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 se- quences shared by as many micro-organisms 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.
  • 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 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, polyhy- droxyethoxylated castor oil, peanut oil, olive oil, gelatine, lactose, terra alba, sucrose, glucose, cyclodext n, 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 monoglycehdes and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hy- droxymethylcellulose 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 fla- vouring 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, intra urethra I, 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.
  • 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 cap- sules 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 carrier 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 mole- cules 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 are used in the experimental part: (The 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.
  • 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)
  • the PNA sequence was H-KKK-T x ACT x CAT x ACTCT-LysNH 2 .
  • the PNA sequence was H-RRR-T x ACT x CAT x ACTCT-LysNH 2 .
  • Anti beta-lactamase activity shows relative ⁇ -lactamase activity in converting nitrocefin. The values are the mean of four replicates with standard deviations indicated. The growth curves at 37 °C (not shown) shows that only PNA 2058 was slightly inhibitory (1 ⁇ M) to growth and only in the K12 strain (meaning that the PNAs were not toxic at the concentration used). The negative results regarding the K12 strain ( Figure 7, 8) prompted us to investigated the anti ⁇ -lactamase activity in the more permeable AS19 strain ( Figure 9, 10). In this case, both at 500 nM and at 1 ⁇ M concentration weak effects were in fact seen.
  • the more lipophilic (2060 and 2063) and the more basic (2058 and 2067) PNAs showed improved anti ⁇ -lactamase activity as compared to the parent PNA (2005). Since the more lipophilic PNAs have low affinity towards RNA they probably excelled their effect by improved passive diffusion into the cells.
  • Boc-D-Hse(OBzl)-OH was from Advanced Chemtech. Boc-D-3-(2-pyridyl)-Ala-OH and Boc-D-3-(4-pyridyl)-Ala-OH were from Synthetech. Boc-D-Phe-OH, Boc-D-Ser(OBzl)- OH, Boc-D-Cit-OH and Boc-D-Arg(di-Z)-OH were from Bachem. Boc-D-Gln-OH, H-D-Leu- OBzl.PTSA, H-D-Ala-OBzl.PTSA were from NovaBiochem. Antisense experiment:
  • Lysine (2-CI-Z) monomer (4c).
  • Cs 2 CO 3 (3.30 g, 10.12 mmol) was added to a stirred solution of Boc-D-Lys(2-CI-Z)-OH (4.00 g, 9.64 mmol) in DMF (35 ml).
  • allyle bromide (1.53 g, 12.65 mmol) was added and the mixture was stirred overnight and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat Na- HCO 3 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml).
  • Citrulline monomer (4e). Cs 2 CO 3 (2.49 g, 7.63 mmol) was added to a stirred solution of Boc-D-Cit-OH (2.0 g, 7.27 mmol) in DMF (26 ml). After 15 min benzyl bromide (1.04 ml, 8.72 mmol) was added and the mixture was stirred for 1.5 hr and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo.
  • Glutamine monomer (4f) Cs 2 CO 3 (5.54 g, 17.05 mmol) was added to a stirred solution of Boc-D-Gln-OH (4.00 g, 16.2 mmol) in DMF (58 ml). After 20 min benzyl bromide (3.58 g, 20.9 mmol) was added and the mixture was stirred for 2 hr and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO 3 (75 ml) was added, and the mixture was extracted with AcOEt (150 ml).
  • Pd(PPh 3 ) 4 (82 mg, 0.0708 mmol) was added to a solution of 3h (3.96 g, 7.08 mmol) and morpholine (6.2 ml, 70.8 mmol) in THF (70 ml). The solution was stirred 30 min at rt, and then evaporated in vacuo. The white foam was dissolved in AcOEt (200 ml) and extracted with 10% citric acid (100 ml). The H2O phase was extracted with AcOEt (2 x 200 ml). The combined organic phases were washed with 10% citric acid (50 ml), brine (50 ml) and dried (Na 2 SO ).
  • Phenylalanine monomer (4i). Cs 2 CO 3 (6.43 g, 19.7 mmol) was added to a stirred solution of Boc-D-Phe-OH (5.00 g, 18.8 mmol) in DMF (67 ml). After 20 min benzyl bromide (3.88 g, 22.6 mmol) was added and the mixture was stirred for 2 hr and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO 3 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml). The organic phase was washed with brine (100 ml), dried over Na 2 SO 4 , and evaporated in vacuo.
  • Boc-D-Phe-OH was obtained by flash chromatography (Eluent: AcOEtHexane 1 :3). Yield 6.42 g, 96%.
  • TFA 14 ml was added at 0 °C to a solution of Boc-D-Phe-OBzl (6.40 g, 18.0 mmol) in CH CI 2 (14 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO 3 (200 ml).
  • Boc-D-4-Py-OBzl was obtained by flash chromatography (Eluent: AcOE Hexane 2:1 ). Yield 5.36 g (80%).
  • TFA (11.6 ml) was added at 0 °C to a solution of Boc-D-4-Py-OBzl (5.35 g, 15.00 mmol) in CH 2 CI 2 (11.6 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO 3 (200 ml).
  • Boc-D-2-Py-OBzl was obtained by flash chromatog- raphy (Eluent: AcOELHexane 1 :1 ). Yield 6.54 g (98%).
  • TFA 14 ml was added at 0 °C to a solution of Boc-D-2-Py-OBzl (6.38 g, 17.9 mmol) in CH 2 CI 2 (14 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO 3 (200 ml).
  • PNA 2060 [TT34 fr 1 1]: MALDI-MS 3474 (calc. 3474).
  • PNA 2066 [TT49 fr 4]: MALDI-MS 3521 (calc. 3519).
  • PNA 2067 [TT51 fr11]: MALDI-MS 3613 (calc. 3609).
  • PNA 2068 [TT53 fr 3]: MALDI-MS 3606 (calc. 3606).
  • PNA 2064 [AP-769V fr 3]: MALDI-MS 3519 (calc. 3519).
  • PNA 2061 PT37 fr 5]:. MALDI-MS 3400 (calc. 3396).
  • PNA 2069 [TT54 fr 4]: MALDI-MS 3438 (calc. 3438).
  • PNA 2063 [AP789 fr 12]: MALDI-MS 3577 (calc. 3576).
  • PNA 2065 [AP-769II fr 2]: MALDI-MS 3576 (calc. 3576).
  • Example 3 Preparation of H-KFFKFFKFFK- ado -TTC AAA, CAT AGT-NH 2
  • the peptide-PNA-chimera H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NH 2 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 solu- tion 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.
  • 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 re- moved 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.
  • PNA-oligomer ado- ⁇ c AAA CAT AGT-NH 2 (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-(/V- 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 re- action mixture was stirred for further 2.5 h. The product was precipitated in diethylether (10 mL).
  • Example 5 Conjugation of peptide and maleimide activated PNA
  • a solution of peptide CKFFKFFKFFK (0.5 mg in 200 ⁇ l degassed Tris buffer 10mM, pH 7.6 (329 nM)) was added to a solution of the above activated product (0.2 mg in 200 ⁇ l DMF:Water 1 :1 ).
  • the reaction mixture was stirred over night.
  • the target compound was purified by HPLC directly from the crude reaction mixture.
  • 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-TCCCTCTC-Lys-NH 2 instead Of ado-TTC AAA CAT AGT-NH 2 .
  • 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, (125).

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Abstract

La présente invention concerne de nouveaux médicaments destinés à combattre des maladies.
PCT/DK2002/000005 2001-01-05 2002-01-03 Molecules pna modifiees WO2002053574A2 (fr)

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WO2021211786A1 (fr) * 2020-04-17 2021-10-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Thyclotides
US11236130B2 (en) 2013-04-11 2022-02-01 Carnegie Mellon University Divalent nucleobase compounds and uses therefor
US11319349B2 (en) 2013-04-11 2022-05-03 Carnegie Mellon University Template-directed PNA synthesis process and PNA targeting compounds
US11603369B2 (en) 2016-09-26 2023-03-14 Carnegie Mellon University Divalent nucleobase compounds and uses therefor

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GB9621367D0 (en) * 1996-10-14 1996-12-04 Isis Innovation Chiral peptide nucleic acids
US6306993B1 (en) * 1997-05-21 2001-10-23 The Board Of Trustees Of The Leland Stanford, Jr. University Method and composition for enhancing transport across biological membranes
JP2002511885A (ja) * 1997-07-24 2002-04-16 ザ パーキン―エルマー コーポレーション 脂質膜を横切る輸送のための膜透過性構築物
HUP0203465A2 (hu) * 1998-11-11 2003-01-28 Pantheco A/S Peptidek és nukleinsav-analóg, mint például PNS, LNS vagy morfolino közötti konjugátumok
US6521456B1 (en) * 1999-01-08 2003-02-18 Gregor Siebenkotten Cellular transport system for the transfer of a nucleic acid through the nuclear envelope and methods thereof
WO2001076636A2 (fr) * 2000-04-06 2001-10-18 Pantheco A/S Composition pharmaceutique a base de molecules de pna modifiees

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11236130B2 (en) 2013-04-11 2022-02-01 Carnegie Mellon University Divalent nucleobase compounds and uses therefor
US11319349B2 (en) 2013-04-11 2022-05-03 Carnegie Mellon University Template-directed PNA synthesis process and PNA targeting compounds
US11713340B2 (en) 2013-04-11 2023-08-01 Carnegie Mellon University Divalent nucleobase compounds and uses therefor
US11603369B2 (en) 2016-09-26 2023-03-14 Carnegie Mellon University Divalent nucleobase compounds and uses therefor
WO2021211786A1 (fr) * 2020-04-17 2021-10-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Thyclotides

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