WO2001027261A2 - Conjugates between a peptides and a nucleic acid analog, such as a pna, lna or a morpholino - Google Patents

Abstract

The present invention concerns novel drugs for use in combating infectious micro-organisms, in particular bacteria. More particularly the invention concerns peptide nucleic acid (PNA) sequences, which are modified by conjugating cationic peptides to the PNA in order to obtain novel PNA molecules with enhanced anti-infective properties.

Description

CONJUGATES BETWEEN A PEPTIDES AND A NUCLEIC ACID ANALOG , SUCH AS A PNA, LNA OR A MORPHOLINO

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.

BACKGROUND OF THE INVENTION

10 From the discovery of penicillin in the 1940's there has been an ever-growing search for new drugs. Many drugs or antibiotics have been discovered or developed from already existing drugs. However, over the years many strains of bacteria have become resistant to one or more of the currently available drugs, which were effective, drugs in the past. The number of antibiotic drugs currently being used by

15 clinicians is more than 100.

Most 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.

25

Because of the emergence of antibiotic resistant bacterial pathogens, there is an ongoing need for new therapeutic strategies. One strategy to avoid problems caused by resistance genes is to develop anti-infective drugs from novel chemical classes for which specific resistance traits do not exist.

30

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

35 these is 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.

Results from basic research have been encouraging, and antisense oligonucleotide drug formulations against viral and disease causing human genes are progressing through clinical trials. Efficient antisense inhibition of bacterial genes also could have wide applications; however, there have been few attempts to extend antisense technology to bacteria.

Peptide nucleic acids (PNA) are compounds that in certain respects are similar to oligonucleotides and their analogs and thus may mimic DNA and RNA. In PNA, the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 (29)) (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

(30)). The Pseudo-peptide backbone provides superior hybridization properties

(Egholm et al. 1993 (30)), resistance to enzymatic degradation (Demidov et al. 1994

(31)) and access to a variety of chemical modifications (Nielsen and Haaima 1997 (32)).

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.

Furthermore, homopyrimidine PNA oligomers form extremely stable PNA₂-DNA triplexes with sequence complementary targets in DNA or RNA oligomers. Finally, PNA's may bind to double stranded DNA or RNA by helix invasion.

An advantage of PNA compared to oligonucleotides is that the 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. As a result, PNA's are resistant to degradation by enzymes unlike nucleic acids and peptides.

For antisense application, 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.

As an antisense agent for microorganisms, 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).

However, none of these disclosures discuss ways of transporting the PNA across the bacterial cell wall and membrane.

Furthermore, for bacterial application, poor uptake is expected, because bacteria have stringent barriers against foreign molecules and antisense oligomer containing nucleobases appear to be too large for efficient uptake. The results obtained by Good and Nielsen (1998a,b (39,40)) indicate that PNA oligomers enter bacterial cells poorly by passive diffusion across the lipid bilayers. 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. However, 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. According to this publication, 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.

Thus, despite the promising results in the use of the PNA technology obtained previously, there is a great need of developing new PNA antisense drugs, which are effective in combating microorganisms.

SUMMARY OF THE INVENTION

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.

Surprisingly, it has been found out that by incorporating a peptide, an enhanced anti-infective effect can be observed. The important feature of the 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.

Thus, the present invention concerns a modified PNA molecule of formula (I):

Peptide - L - PNA (I) wherein L is a linker or a bond;

Peptide is any amino acid sequence and

PNA is a Peptide Nucleic Acid, and pharmaceutically acceptable salts thereof.

More particularly, the present invention concerns a modified PNA molecule of formula (I)

Peptide - L - PNA (I)

wherein Peptide is a cationic peptide or cationic peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the formula (II):

C-(B-A)_n-D, (II)

Wherein 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.

In one embodiment, 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. In a preferred embodiment of the invention the peptide contains from 2 to 18 amino acids, most preferred from 5 to 15 amino acids.

In another preferred embodiment of the invention 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. In another embodiment, 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.

In a preferred embodiment of the invention 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.

In a preferred embodiment the peptide is linked to the PNA sequence via the carboxy end.

Within the present invention, 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. Examples of such 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. In the alternative, 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.

In another aspect of the invention the modified PNA molecules are used in the manufacture of medicaments for the treatment or prevention of infectious diseases or for disinfecting non-living objects.

In a further aspect, the invention concerns a composition for treating or preventing infectious diseases or disinfecting non-living objects.

In yet another aspect, the invention concerns the treatment or prevention of infectious diseases or treatment of non-living objects.

In yet a further aspect, 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.

In yet a further aspect, 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.

PCT Publication WO99/14226, WO98/03533 and WO98/32467 are all incorporated by reference.

Thus, further preferred compounds of the invention are modified oligonucleotides of the formula (III):

Peptide - L - Oligon (III)

wherein L is a linker or a bond; Peptide is any amino acid sequence and Oligon designates an oligonucleotide or analogue thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

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

DETAILED DESCRIPTION OF THE INVENTION 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.

Without being bound by theory, it is believed that 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. However, an improved accessibility to the nucleic acid target or an improved binding of the PNA may also add to the overall effect observed.

According to the invention, 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.

Accordingly, the present invention concerns novel modified PNA molecules having the formula:

Peptide - L - PNA, wherein

L is a linker or a bond;

PNA is a peptide nucleic acid sequence; and Peptide is a cationic peptide or peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the formula:

C-(B-A)_n-D, wherein

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.

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.

By the terms "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. Similarly the term "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.

Among the positively charged amino acids and amino acid analogs may be mentioned lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and ornithine (Orn). The skilled person will be aware of further positively charged amino acids and amino acid analogs.

Among the 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, β-cyclohexylalanine, 4- chlorophenylalanine, norleucine and phenylglycine. The skilled person will be aware of further non-charged amino acids and amino acid analogs.

Preferably, 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 β-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.

Other preferred Peptides are 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), and any subunits thereof comprising at least 3 amino acids whereof at least one amino acid is a positively charged amino acid.

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-NH₂ (SEQ ID NO: 158) as well as β-amino-acid oligomers (β-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. On the other hand, 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. Preferably, 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 are described in WO 96/11205 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-(Λ/-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-ε- maleimido-caproylate), SMPH (succinimidyl 6-(β-maleimido-propionamido) hexanoate, AMAS (succinimidyl N-(α-maleimido acetate), SMPB (succinimidyl 4-(p- maleimidophenyl)butyrate), β.ALA (β-alanine), PHG (Phenylglycine), ACHC (4- aminocyclohexanoic acid), β.CYPR (β-(cyclopropyl) alanine) and ADC (amino dodecanoic acid).

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.

In a preferred embodiment the linking group is a combination of the β.ALA linking group or the ADO linking group with any of the other above mentioned linking groups.

Thus, preferred linkers are -achc-β.ala-, -ache-ado-, -Icsmcc-β.ala-, -mbs-β.ala-, - emcs-β.ala-, -lcsmcc-ado-, -mbs-ado-, -emcs-ado- or -smph-ado-.

Most preferred are the linkers -achc-β.ala-, -lcsmcc-ado- and -mbs-ado-.

In the case SMCC (succinimidyl 4-(Λ/-maleimidomethyl)cyclohexane-1-carboxylate ) is used in the process of linking PNA to the peptide, it is necessary to add a cysteine (C) or a similar thiol containing moiety to the terminal end of the peptide (see Fig. 2). Additionally, amino acids, such as glycine, may be a part of the linker.

The chemical structures of the different succinimidyl based linking groups used in the conjugation of the Peptide and PNA is shown in Figure 5.

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 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. Alternatively, the target may be a gene encoding a factor responsible for resistance to antibiotics. In a preferred embodiment, 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. As used in the present invention, 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. In a preferred embodiment, PNA targeted to single strand RNA is complementary in an anti-parallel orientation.

In a another preferred embodiment of the invention 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 PNA₂-RNA (PNA₂-DNA) triple helix.

In another preferred embodiment of the invention, 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.

In a preferred embodiment of the PNA the backbone is aminoethylglycine as shown in Figure 1.

Potential 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.

A further consideration is that some physiological processes are primarily active in dividing cells whereas others are running under non-dividing circumstances as well.

Known target proteins in cell wall biosynthesis are penicillin binding proteins, PBPs, 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. Only the high molecular weight PBPs are known to be essential for growth and have therefore been chosen as targets for PNA antisense. Protein biosynthesis is an important process throughout the bacterial cell cycle. Therefore, the effect of targeting areas in the field of protein biosynthesis is not dependent on cell division.

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).

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) 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 10⁴ 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.

Examples of 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,

The incidence of the multiple antimicrobial resistance of bacteria which cause infections in hospitals/intensive care units is increasing. These include methicillin- resistant and methicillin-vancomycin-resistant Staphylococcus aureus, vancomycin- resistant enterococci such as Enterococcus faecalis and Enterococcus faecium, penicillin-resistant Streptococcus pneumoniae and cephalosporin and quinolone resistant gram negative rods (conforms) such as E. coli, Klebsiella pneumoniae, Pseudomonas species and Enterobacter species. More recently, pan antibiotic (including carbapenems) resistant gram negative bacilli have emerged. The rapidity of emergence of these multiple antibiotic-resistance is not being reflected by the same rate of development of new antibiotics and it is, therefore, conceivable that patients with serious infections soon will no longer be treatable with currently available antimicrobials (1 , 2). Several international reports have highlighted the potential problems associated with the emergence of antimicrobial resistance in many areas of medicine and also outlined the difficulties in the management of patients with infections caused by these micro-organisms (3, 20).

A. Gram positive bacteria

Methicillin-resistant S. aureus (MRSA) (4,5), methicillin-vancomycin resistant S. aureus (VMRSA) and vancomycin resistant enterococci (VRE) have emerged as major nosocomial pathogens (3, 6, 7, 18). Vancomycin is currently the most reliable treatment for infections caused by MRSA but the potential transfer of resistance genes from VRE to MRSA may leave few therapeutic options in the future.VRE , as well as providing a reservoir of vancomycin resistance genes, can also cause infections in patients with compromised immunity, which are difficult to treat, with some strains showing resistance to all major classes of antibiotic. The increasing incidence of VRE strains among clinical isolates of enterococci places them as important nosocomial pathogens and in some hospitals in the United States VRE are responsible for more than 20% of enterococcal infections (17, 18)

S aureus showing intermediate vancomycin resistance (VISA) as well as VMRSA have now been reported from several numbers of centres/hospitals worldwide (8, 9). Of the S. aureus isolates from USA, Europe and Japan 60 -72% were MRSA and most strains being multi-drug-resistant MRSA are the commonest cause of surgical site infection and comprise 61 % of all such S. aureus infections and a major cause of increased morbidity and mortality of ICU patients (21 , 22, 23,19).

Coagulase negative staphylococci (CNS) 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)

B. Gram negative bacteria Pseudomonas aeruginosa, Pseudomonads species including Burkholderia cepacia and Xanthomonas malthophilia, Enterobacteriaceae including E. coli, Enterobacter species and Klebsiella species account for the majority of isolates where resistance has emerged (25,26, 3)

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%.

Selective pressure through the use of broad-spectrum antibiotics has lead to multidrug resistance in Gram-negative bacteria. Each time a new drug is introduced, resistant subclones appear and today the majority of isolates are resistant to at least one antimicrobial ( 20, 14, 25, 26 )

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)

Several international reports have highlighted the potential problems associated with the emergence of antimicrobial resistance in bacteria mentioned above, and it is, therefore, conceivable that patients with serious infections soon will no longer be treatable with currently available antimicrobials. The increasing incidence of resistant strains among clinical isolates of S.aureus, S.epidermidis (CNS), enterococci, Streptococcus pneumoniae, gram negative bacilli (coliforms) such as E.coli, Klebsiella pneumoniae, Pseudomonas species and Enterobacter species make these bacteria major candidates for future PNA design.

METHODS 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. For the purpose of exemplifying the present invention, 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.

To illustrate one example of measuring growth and growth inhibition the following procedure may be used:

Bacterial strain: E.coli K12 MG1655

Media: 10% Mueller-Hinton broth, diluted with sterile water.

10% LB broth diluted with sterile water. 100% Mueller-Hinton broth. Trays: 96 well trays, Costar # 3474, Biotech Line AS, Copenhagen. (Extra low sorbent trays are used in order to prevent / minimize adhesion of PNA to tray surface).

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 5x10⁵ and 5x10⁴ bacteria/ml medium in each well, containing 200 μl 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.

Growth inhibitory effect of PNA-constructs: 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.

Total inhibition of bacterial growth is defined as: OD (16h) = OD (Oh) or no visible growth according to NCCLS Guidelines

In an initial screening the 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)).

In vivo antibacterial efficacy is established by testing a compound of the invention in the mouse peritonitis/sepsis model as described by N. Frimodt-Møller et al. 1999, Chap. 14, Handbook of Animal Models of Infection.

For the in vivo efficacy experiment a number of female NMRI mice are inoculated with approximately 10⁷ cfu of E. coli ATCC 25922 intraperitoneally. Samples are drawn from blood and peritoneal fluid at 1 , 2, 4 and 6 hrs post infection, and cfu/ml counted. 1 hr post infection the animals are treated once in groups with: 1. Gentamicin (38 mg/kg s.c); 2. Ampicillin (550 mg/kg s.c); 3. a compound of the invention (50 - 60 mg/kg i.v.); 4. no treatment.

In another aspect of the present invention, 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.

In a further aspect of the present invention, 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. In one embodiment, 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. In another embodiment, 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. In the latter form, specific combinations of target bacteria may be selected to the treatment. Alternatively, the target can be one or more genes, which confer resistance to one or more antibiotics to one or more bacteria. In such a treatment, the composition or the treatment further comprises the use of said antibiotic(s).

In another aspect, 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.

Pharmaceutical 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.

Typical 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. In making the compositions, conventional techniques for the preparation of pharmaceutical compositions may be used. For example, 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. 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. Some examples of 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. Similarly, 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.

The pharmaceutical 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.

If a solid carrier is used for oral administration, 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. If 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.

For nasal administration, the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. 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. For parenteral application, particularly suitable are 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.

In formulations for treatment or prevention of infectious diseases in mammals 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.

Usually, 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.

In a still further aspect, 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.

In yet another aspect of the present invention, 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. Such a treatment may be in the form of administering a composition in accordance with the present • invention. In particular, 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.

In yet a further aspect of the present invention, 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.

EXAMPLES

The following examples are merely illustrative of the present invention and should not be considered limiting of the scope of the invention in any way. The principle of the present invention is shown using E. coli as a test organism. However, as shown in Example 19, the advantageous effect applies in the same way to other bacteria.

The following abbreviations related to reagents are used in the experimental part: (The monomers and the PNA sequences are stated in bold)

25

The following abbreviations relating to 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.)

The 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 μm, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.

Mass Spectrometry was performed on MALDI (Matrix Assisted Laser Desorption and lonisation Time of Flight Mass Spectrometry) as HP MALDI-TOF # G2025A calibrated with peptide nucleic acids of the following weights:

= 1584.5 g/mol, Mw₂ = 3179.0 g/mol and Mw₃ = 4605.4 g/mol.

Example 1

Preparation of H-KFFKFFKFFK-ado-ττc AAA CAT AGT-NH_? (SEQ ID NO: 18) The peptide-PNA-chimera H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 18) was synthesized on 50 mg MBHA resin (loading 100 μmol/g) (novabiochem) in a 5 ml glass reactor with a D-2 glassfilter. Deprotection was done with 2x600 μL TFA/m-cresol 95/5 followed by washing with DCM, DMF, 5% DIEA in DCM and DMF. The coupling mixture was 200 μl 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 μl 0.5 M DIEA in pyridine and activated for 1 min with 200 μl 0.202 M HATU (PE- biosystems) in NMP. The coupling mixture for the peptide part was 200 μl 0.52 M NMP solution of amino acid (Boc-Phe-OH and Boc-Lys(2-CI-Z)-OH (novabiochem)) mixed with 200 μl 1 M DIEA in NMP and activated for 1 min with 200 μl 0.45 M HBTU in NMP. After the coupling the resin was washed with DMF, DCM and capped with 2 x 500 μl 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 μm, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B. Mw calculated: 4791.9 g/mol; found on MALDI: 4791 g/mol.

Example 2

Maleimide activation of PNA

PNA-oligomer ado-ττc AAA CAT AGT-NH₂ (SEQ ID NO: 19) (purified by HPLC) (2 mg, 0.589 μmol, Mw 3396.8) was dissolved and stirred for 15 min in NMP:DMSO 8:2 (2 ml). Succinimidyl 4-(Λ/-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (PIERCE)(1.1 mg, 3.24 μmol, 5.5 eq.) dissolved in NMP (50 μl) and DIEA (34.7 μl, 198.7 μmol) 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.

Example 3

Conjugation of peptide and maleimide activated PNA A solution of peptide CKFFKFFKFFK (SEQ ID NO: 20) (0.5 mg in 200 μl degassed Tris buffer 10mM, pH 7.6 (329 nM)) was added to a solution of the above activated product (0.2 mg in 200 μl 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 μm, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B. Mw calculated: 5133.0 g/mol; found on MALDI: 5133 g/mol.

Example 4 H-LLKKLAKALKG-a ex-ado-CCATCTAATCCT-NH₂ (SEQ ID NO: 21)

Performed in accordance with example 1 , however with the use of 6-aminohexanoic acid (ahex) as linker together with 8-amino-3,6-dioxaoctanoic acid (ado).

Example 5

Preparation Of H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCCCTCTC-Lys-

NH, (SEQ ID NO: 22)

Performed in accordance with example 1 , however with the use of PNA oligomer ado-JTJTJJT-ado-ado-ado-τcccτcτc-Lys-NH₂ (SEQ ID NO: 23) instead of ado-ττc AAA CAT AGT-NH₂ (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)).

Example 6

Preparation of peptide-PNA-chimeras Different peptide-PNA-chimeras were prepared in the same way as described above.

Example 7

The peptide-PNA-chimeras in Table I were prepared as described in Example 1 using the linking groups as defined above:

Table

Example 8

The peptide-PNA-chimeras in Table III were prepared as described in Example 1 using the linking groups as defined above.

Table III

Example 9

Description of a primary screen

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.

Components

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 H₂0 as a 40x concentrated stock solution.

Assay conditions From an over night culture a fresh culture (test culture) is grown to mid-log-phase (OD₆oo = 0.1 corresponding to 10⁷ cells/ml) at 37 °C. The test culture is diluted stepwise in the range 10^s to 10¹ with 10% LB medium. 195 μl of diluted cultures plus 5 μl 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. OD₆oo measurements are performed automatically every 3.19 minutes and recorded simultaneously.

Target genes:

Penicillin binding proteins (PBPs)

PBPs act in biosynthesis of murein (peptidoglycan), which is part of the envelope of Gram-positive and Gram-negative bacteria. 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. PNA design no. 1

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:

sense 5 ' AATGGGAAATTTCCAGTGAAGTTCGTAAAG 3 ' (SEQ ID NO: 142)

121 + + + 150 antisense 3 ' TTACCCTTTAAAGGTCACTTCAAGCATTTC 5 ' (SEQ ID NO: 143)

Both the coding and the non-coding (antisense) strand of the GTG start codon region are shown.

The sequence of the GTG start codon region of the antisense strand and PNA26 are shown in the 5' to 3' orientation:

antisense 5' CTTTACGAACTTCACTGGAAATTTCCCATT 3' (SEQ ID NO: 143) PNA26 H-KFFKFFKFFK-ado-CACTGGAAATTT-Lys-NH₂ (SEQ ID NO: 144)

PNA26 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide.

Growth assay with PNA26 The assay was performed as follows:

Dilutions of the test culture corresponding to 10⁵, 10⁴, 10³, 10² and 10¹ cells/ml containing PNA26 at a final concentration of 1.5, 2.0, 2.5, 3.0 and 3.5 μM are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with 10⁴-10¹ cells/ml and a PNA concentration of at least 2.5μM (Table 1).

PNA design no. 2

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:

sense 5 ' GAGTAGAAAACGCAGCGGATGAAACTACAGAAC 3 ' (SEQ ID NO: 145)

99 + _{+ + 131} antisense 3' CTCATCTTTTGCGTCGCCTACTTTGATGTCTTG 5' (SEQ ID NO: 146)

Both the coding (sense) and the non-coding (antisense) strand of the GTG start codon region are shown.

In the following sequence of the ATG start codon region of the antisense strand and PNA26 are shown in the 5' to 3' orientation:

antisense 5' GTTCTGTAGTTTCATCCGCTGCGTTTTCTACTC 3' (SEQ ID NO: 146) PNA14 HKFFKFFKFFK-ado-TTTCATCCGCTG-Lys-NH₂ (SEQ ID NO: 147)

PNA14 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide.

Growth assay with PNA14 The assay was performed as follows:

Dilutions of the test culture corresponding to 10⁵, 10⁴, 10³, 10² and 10¹ cells/ml containing PNA14 at a final concentration of 1.3, 1.4 and 1.5 μM are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with 10⁴-10¹ cells/ml and a PNA concentration of at least 1.4μM (Table 2).

Example 10

Bacterial growth inhibition with PNA against the LacZ gene.

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 - NH₂ terminal lysine.

The assay was performed as follows:

Dilutions of the test culture E. coli K12 corresponding to, 5x10⁵ and 5x10⁴, 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.

Total inhibition of growth can be seen in cultures with 5x10 -10⁵ cells/ml and a PNA concentration of at least 300nM (see Table 3). The results show that the basic KFF motif 10-mer as well as truncated peptides thereof (4, 5, 6, and 9mer) may be used to enhance the inhibitory effect of PNA.

Table 1 Bacterial growth inhibition with PNA 26; E. coli K12 in 10% Mueller-Hinton broth.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times nd: Not done O CD

PNA PNA cone, in wells nM

1300 1400 1500

Bacterial 1% 0.1% 0.1% 0.001% 1% 0.1% 0.1 % 0.001% 1% 0.1% 0.1% 0.001% concentration 0.0001% 0.0001% 0.0001%

14 - (+) + + + + + + +

+ +

Table 2 Bacterial growth inhibition with PNA 14; E. coli K12 in 10% Mueller-Hinton broth.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times. nd: Not done

O

TABLE 3.

+ : Total inhibition of bacterial growth.

+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times;

Nd: Not done

Example 11

Bacterial growth inhibition with PNA against the infA gene of E. coli (seguence as PNA 130).

The PNA130 and 218-226 against the infA-gene have been synthesized with peptides as truncated versions of the KFF-motif.

Growth assay with PNA130 The assay was performed as follows:

Dilutions of the test culture E. coli K12 corresponding to, 2x10⁴ and 4x10⁴, 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.

Total inhibition of growth can be seen in cultures with 4x10⁴-2x10⁴ cells/ml and a PNA concentration of at least 600nM (Table 4). The results show that the basic KFF motif 10-mer as well as truncated peptides thereof (6 and 9mer) may be used to enhance the inhibitory effect of PNA.

Example 12

Bacterial growth inhibition with PNA against the α-sarcine loop of ribosomal RNA. The PNA's 140-146 against the α-sarcine loop of ribosomal RNA has been synthesized with peptides as truncated versions of the KFF-motif.

Growth assay

The assay was performed as follows:

Dilutions of the test culture E. coli K12 corresponding to, 2x10⁴ and 4x10⁴, cells/ml containing truncated versions of the KFF-motif of the PNA's against α-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.

Total inhibition of growth can be seen in cultures with 5x10⁵-5x10⁴ cells/ml and a PNA concentration of at least 200nM (Table 5). The results show that the basic KFF motif 10-mer as well as all truncated peptides thereof comprising at least 3 amino acids may be used to enhance the inhibitory effect of PNA.

Example 13

Bacterial growth inhibition with PNA against the FtsZ gene of E. coli K12.

Growth assay with PNA170-179 and 109 The assay was performed as follows:

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.

Total inhibition of growth can be seen in cultures with 350-700 cells/ml and a PNA concentration of at least 300nM (Table 6). When comparing 109 with 179, the smcc linker appears to add some advantages to the molecule. Further, sequence 174 shows promising results.

CO

TABLE 4.

+ : Total inhibition of bacterial growth (+): Significantly extended lagphase, (more than five times)

Lagphase extended less than five times nd: Not done

TABLE 5.

+ : Total inhibition of bacterial growth (+): Significantly extended lagphase, (more than five times)

Lagphase extended less than five times nd: Not done

TABLE 6. + : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times) Lagphase extended less than five times; nd: Not done

Example 14

Bacterial growth inhibition with PNA, with different kinds of linker and peptide, against the gene encoding IF-1 of E. coli.

E. coli K12 in 100% Mueller-Hinton broth.

For the 7 PNA's in this set-up, the sequence of the nucleobases is the same as the sequence in PNA 130, but the linking groups and the peptides varies.

Experimental set-up corresponds to the set-up as described in Example 13.

As can be seen from Table 7a and 7b, in the present combination of PNA and Peptide, the smcc-ado linker seems to be the superior linker showing total inhibition of growth in cultures with 1.6x10³-8x10² cells/ml and a PNA concentration of at least 600nM.

Example 15

Bacterial growth inhibition with 9 mer peptide

In order to test the effect of the Peptide without the PNA, 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:

Dilutions of the test culture corresponding to 10⁵, 10⁴, and 10³ cells/ml containing the peptide no. 2339 at a final concentration of 100 to 20.000 nM, are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with 7.9x10³ cells/ml and a peptide concentration of at least 20.000 nM, minimal signs of inhibition of growth can be detected at concentrations from 5000 nM (10% medium: Table 8; 100% medium: Table 9). Conclusions: Peptides are active alone but only at very high concentrations and above the range used for PNA growth assays.

Example 16

Bacterial growth inhibition with 9 mer peptide and non-sense PNA

Growth assay of the peptide no. 2339 together with nonsense PNA 136

The assay was performed as follows:

Dilutions of the test culture corresponding to 10⁵, 10⁴, and 10³ cells/ml containing

PNA 136 alone or PNA 136 and the peptide no. 2339 in equal amounts at a final concentration of 400 to 1000 nM, are incubated at 37°C for 16 hours with constant shaking. No inhibition of growth was detected in any of the concentrations (Table

10). Conclusions: nonsense PNA is not active in the chosen range.

TABLE 7.a Data from 100% MH

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times; nd: Not done

D

TABLE 7b. data from 10 % MH

+ : Total inhibition of bacterial growth; (+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times; nd: Not done

n o

TABLE 8. + : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times) ((+)): Lagphase extended less than five times, but still with growth curve effect

Lagphase extended less than five times; nd: Not done

n

TABLE 9. + ; Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

((+)): Lagphase extended less than five times, but still with growth curve effect

Lagphase extended less than five times nd: Not done

TABLE 10.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times) ((+)): Lagphase extended less than five times, but still with growth curve effect

Lagphase extended less than five times; nd : Not done

Example 17

Bacterial growth inhibition with PNA (without peptide) against the gene encoding FtsZ of E. coli and a peptide E. coli K12 in 100% Mueller-Hinton broth.

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-NH₂ (SEQ ID NO: 156). The peptide is also used for PNA 174.

In the wells with both PNA and peptide there is equal amount PNA and peptide.

As can be seen in Table 11 , neither 249 nor 250 alone nor 249 and 250 together show any useful effect in the low concentration end. Only the peptide alone in concentrations above 2500 nM may show growth inhibition effect.

Example 18

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.

From Table 12 it can be seen that the orientation of the Peptide is not so important. However, for specific combinations of PNA and Peptide, one of the orientations may be preferred.

cn

TABLE 11.

+ : Total inhibition of bacterial growth.

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times; nd: Not done

e e

TABLE 12.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times nd: Not done

Example 19

Inhibition of bacterial growth by PNA-peptide with specificity for the ribosomal α- sarine loop In order to show that the present invention may be used in a treatment of many micro-organisms, a selection of Gram-negative and Gram-positive bacteria were treated under the same assay conditions as used in example 12. The modified PNA molecule used is PNA 146.

Gram-negative organisms Inhibition of growth

Escherichia coli +

Klebsiella pneumonia + Pseudomonas aeruginosa + Salmonella typhimurium +

Gram-positive organisms

Staphylococcus aureus + Enterococcus faecium +

Micrococcos luteus +

Conclusions: All of the bacterial isolates were inhibited. Using the same assay conditions used for testing of E. coli K.12, we have demonstrated growth inhibition of different Gram-negative and Gram-positive organisms.

Example 20

Preparation of peptide-PNA-chimeras A peptide-PNA-chimera was prepared in the same way as described in Example 1:

H₂N-SILAPLGTTLVKKVATTLKKI FSK KC-smcc-Ado-TTCTAACATTTA-NH₂ (SEQ

ID NO: 159) . Example 21

Gene target selection and Bacterial growth inhibition with PNA

a. Gene target selection in E. faecalis/E. faecium

The annotated E. faecium genome is, alongside with 250 other genomes, commercially available from Integrated Genomics, Chicago.

Single annotated genes from both organisms are also available in Genbank.

b. In vitro experiments.

The ability of 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 1x10⁴ bacteria/ml medium in each well, containing 195 μl 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.

MIC and MBC:

In addition experiments were carried out to evaluate the relationship between MIC's and MBC's (Minimal Bactericidal Concentration) of the PNA.

The studies were performed on 3 strains of Enterococcus faecium obtained from American Type Culture Collection (ATCC). These strains served as initial indicators of possible interference from known in vivo selected vancomycin resistance mechanisms. The table below summarizes the characteristics of the strains. E. facium Strain Description

8803 susceptible to vancomycin .ciprofloxacin, gentamycin, rifampin, teicoplanin

ATCC 51550 Multidrugresistant (ampicillin, ciprofloxacin, gentamycin, rifampin, teicoplanin, vancomycin)

ATCC 700221 resistant to vancomycin

Experimental setup : MIC's was detected as previously described. Trays were incubated at 35 ° C for further 24 h in order to analyze regrowth of inhibited bacteria (MBC's).

PNA conjugate from Example 20: Bacterial strains: 8803, 51550, 700221 PNA concentration in wells: 400, 800 and 1600 nM

Results

The Minimal Inhibitory Concentrations (MIC^'s) of the PNA conjugate were as follows:

Example 22

Preparation of peptide-PNA-chimeras

A peptide-PNA-chimera was prepared in the same way as described in Example 1 : H₂N-KKFKVKFWKKC-smcc-Ado-ACTTTGTCGCCC-NH₂ (SEQ ID NO: 160) .

Example 23

Gene target selection and Bacterial growth inhibition with PNA

The selection of potential gene targets and testing of ensuing PNA constructs have been performed with 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.

a. Target selection approach

The basic approach used was similar to that used in the previous example. Potential target genes were retrieved from the unfinished genome sequences of S. aureus at the OU-ACGT as well as Genbank. The presence of homologous genes and target sequences in bacterial genomes were tested by using the BLAST 2.0 programs at the NCBI (National Center for Biotechnology Information) www BLAST server.

The antibacterial PNA conjugate prepared in Example 22 was used for the following experiments:

b. In vitro experiments

The ability of 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 1x10⁴ bacteria/ml medium in each well, containing 195 μl 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.

MIC and MBC:

In addition experiments were carried out to evaluate the relationship between MIC's ( Minimal Inhibitory Concentration) and MBC's (Minimal Bactericidal Concentration) of the PNA^'s.

The studies were performed on the reference strain Staphylococcus aureus NCTC 8325 obtained from Prof. J. landolo, University of Oklahoma Health Sciences Center, Department of Microbiology and Immunology. In addition we included two vancomycin resistant isolates of S.aureus obtained from American Type Culture Collection. These strains served as initial indicators of possible interference from known in vivo selected vancomycin resistance mechanisms. The table below summarizes the characteristics of the strains.

S.aureus Strain Description Vancomycin MIC (μg/ml)

8325 Susceptible to methicillin, vancomycin < 0.5

ATCC 700698 Intermediate vancomycin resistant. Resistant to methicillin

ATCC 700698R highly vancomycin resistant subclone of 11 ATCC 700698 Experimental setup :

MIC's were detected as described above. Trays were incubated at 35 ° C for further

24 h in order to analyze regrowth of inhibited bacteria (MBC's).

PNA from Example 22:

Bacterial strains: 8325, 700698, 700698R

PNA concentration in wells: 400, 800 and 1600 nM

Results The Minimal Inhibitory Concentrations (MIC) were as follows:

Example 24

A compound of the invention was tested for antibacterial effect in vivo according to the test described by N. Frimodt-Møller.

Untreated animals developed fulminant clinical signs of infection. At all time points the compound of the invention suppressed the E. coli cfu/ml compared to non- treated controls and was as efficient as the two positive controls. References relating to the Survey of antimicrobial resistance:

I . 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

3. House of Lords Select Committee on Science and Technology. Resistance to antibiotics and other antimicrobial agents. London: HMSO, 1998

4. Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev; 10: 781-91

5. Elliott TSJ. Methicillin-resistant S. aureus (MRSA) and its impact on surgery. Current Medical Literature-Surgical Infections 9(1), January 1997.

6. Arthur M, reynolds PE, Courvalin P. Glycopeptide resistance in enterococci. Agents with novel mechanisms and sites of action. Trends Microbiol 1996; 4: 410-7

7. Zervos M. Vancomycin-resistant Enterococcus faecium in the ICU and Quinopristin/dalfopristin. New 1996; 4: 385-92

8. Johnson AP. Intermediate vancomycinresistance in S. aureus: a major threat or a monor inconvenience? J Antimicrob Chemother 1998; 42: 289- 91.

9. Hiramatsu K, Aritaka N, Hanaki H, Kavasaki S et al. Dissimination in Japanese hospitals of strains of S. aureus heterogeniously resistant to vancomycin. Lancet 1997; 350: 1670-3.

10. Baquero F. Pneumococcal resistance to betalactam-antibiotics : a blobal overview. Microb Drug Resist 1995; 1 : 115-20.

I I . Chandrasekar PH, Kruse JA. Mathews MF. Nosocomial Infection among patients in different types of intensive care units at a city hospital. Crit Care Med 1980-15: 508-10

12. Northey D, Adress ML, Hartsuck JM, Rhoades ER. Microbial surveillance in a surgical intensive care unit. Surg Gynaecol Obstet 1974; 139: 321-5.

13. Vincent J-L, Bilhari DJ, Suter PM at al. The prevalence of nosocomial infection in ICU in Europe. LAMA 1995; 27: 639-44

14. Giwercman B., Labert P, et al. Rapid emergence of resistance in P. aeruginosa in cystic fibrosis patient due to in vivo selection of stable partially derepressed betalactamase producing strains. J Antimicrob Chemother. 1990; 26: 247-259.

15. Hsueh PR, Teng L-J, Yang P-C, Chen Y-C, Ho S-W, Luh K-T. Persistence of a multidrug-res/s ant Pseudomonas aeruginosa clone in an intensive care burn unit. J Clin Microbiol 1998; 36. 1347-51

16. Bert N, Lambert-Zechovsky N. Comparative distribution of resistance patterns and serotypes in Pseudomonas aeruginosa isolates from intensive care units and other wards. J Antimicrob Chemother 1996; 37: 809-13

17. Mcneeley DF, Brown AE, Noel GJ. Chung M, de Lencaster H. An investigation of vancomycin -resistant Enterococcus faecium within the pediatric service of a large urban medical center. Pediatr Infect Dis J 1998; 17: 184-8

18. Carmelli Y, Samore MH, Huskins C.The Association Between Antecedent Vancomycin treatment and Hospital-acquired Vancomycin - Resistant Enterococci. Arch Intern Med 1999; 159: 2461-2468

19. Richards MJ, Edwards JR et al. Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance system. Crit Care Med 1999; 5: 887-92

20. Lepellier D, Caroff N, Reynaud A, Richet H. E. coli: Epidemiology and analysis of risk factors for infections caused by resistant strains. Clin Infect

Dis 1999; 3: 548-52

21. Communicable Disease Report (CDN) 1999; 9: 8.

22. Cookson B.D. Nosocomial antimicrobial resistance surveillance. J Hosp Infec 1999; 97-103 23. Liu CC, hor LI, Wu YH, huang AH et al. Investigation and elimination of epidemic methicillin-resistant S. aureus in a neonatal intensive care unit.

Chong Hua Min Kuo Hsiao Erh Ko I Hsueh Hui Tsa Chih 1993; 34: 285-93. 24. Costerton el al: Bacterial biofilms in nature and disease. Ann rev Microbiol.

1987; 41 : 435-464. 25. Livermore D. Multiresistance and superbugs. Commun Dis Public Health

1998; 1 : pp 74-6.

26. Livermore DM. Acquired carbapenases. J Antimicrob Chemother 1997; 39: 673-6.

27. Dowell SF. The Best Treatment for Pneumonia. Arch Intern Med 1999; 159: 2461-2468. 28. Communicable Disease Report (CDN) 1999; 10: 7.Baquero F. Pneumococcal resistance to beta-lactam antibiotics: A Global Overview. Microb Drug Resist 1995; 1 :115-20.

29. Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O. Science (1991) 254, 1457-1500.

30. Egholm, M, Buchardt, O, Christensen, L, Behrens, C, Freier, S. M. Driver, D.A., Berg, R.H., Kim, S.K., Norden, B. and Nielsen, P.E. Nature (1993) 365, 566-568.

31. Demidov, V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, O. Sόnnichsen, H. S. and Nielsen, P.E. Biochem. Pharmacol.

(1994) 48, 1310-1313.

32. Nielsen, P.E. and Haaima, G. Chemical Society Reviews (1997) 73-78.

33. Hanvey et al. Science (1992) 258,1481-5.

34. Knudsen, H. and Nielsen, P.E. Nucleic Acids Res. (1996) 24, 494-500. 35. Lewis, L.G. et al. Proc. Natl. Acad. Sci. USA (1996) 93, 3176-81.

36. Meyer, O. et al. J. Biol. Chem. (1998) 273, 15621-7.

37. Nyce, J.W. and Metzger, W.J. Nature (1997) 385 721-725.

38. Pooga, M. et al, Nature Biotechnology (1998) 16, 857-61.

39. Good, L. & Nielsen, P.E. Proc. Natl. Acad. Sci. USA (1998) 95, 2073-2076. 40. Good, L. & Nielsen, P.E. Nature Biotechnology (1998) 16, 355-358.

41. Broome-Smith et al. 1985, Eur J Biochem 147:437-46.

42. Egholm, M.; Dueholm.K. L; Buchardt, 0.;Coull, J.; Nielsen, P. E.; Nucleic Acids Research 1995, 23,217-222.

Claims

1. A modified oligonucleotide of formula (III): Peptide - L - Oligon (III) wherein L is a linker or a bond;

Peptide is any amino acid sequence and

Oligon designates an oligonucleotide or analogue thereof.

2. A compound of claim 1 wherein Oligon is an oligomer of Locked Nucleoside Analogues (LNA) or an oligonucleotide analogue with morpholino backbone.

3. A modified PNA molecule of formula(l): Peptide - L - PNA (I) wherein L is a linker or a bond;

Peptide is any amino acid sequence and

PNA is a Peptide Nucleic Acid, and a pharmaceutically acceptable salt thereof.

4. A modified Peptide Nucleic Acid (PNA) molecule having the formula: Peptide - L - PNA (I) wherein L is a linker or a bond;

PNA is a peptide nucleic acid sequence; and

Peptide is a cationic peptide or peptide analogue or a functionally similar moiety, the peptide or peptide analogue having the formula (II): C-(B-A)_n-D, (II) wherein 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, and a pharmaceutically acceptable salt thereof.

5. A modified oligonucleotide or PNA molecule according to any of the claims 1 to 4 wherein L is a linker comprising one or more -ado- (8-amino-3,6-dioxaoctanoic acid), -smcc- (succinimidyl 4-(Λ/-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- (succini idyl N-ε-maleimido-caproylate), -smph- (succinimidyl 6-(β-maleimido- propionamido) hexanoate, -amas- (succinimidyl N-(α-maleimido acetate), -smpb- (succinimidyl 4-(p- maleimidophenyl)butyrate), -β.ala- (β-alanine), -phg- (Phenylglycine), -ache- (4-aminocyclohexanoic acid), β.-cypr- (β-(cyclopropyl) alanine) and -adc- (amino dodecanoic acid) or any combinations thereof.

6. A modified oligonucleotide or PNA molecule according to claim 5 wherein the linking group is a combination of the -β.ala- linking group or the -ado- linking group with any of the other linking groups as defined in claim 5.

7. A modified oligonucleotide or PNA molecule according to claim 6 wherein the linking group is a combination selected from: -achc-β.ala-, -ache-ado-, -lcsmcc- β.ala-, -mbs-β.ala-, -emcs-β.ala-, -lcsmcc-ado-, -mbs-ado-, -emcs-ado- or -smph- ado-.

8. A modified PNA molecule according to claim 4 to 7, 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.

9. A modified PNA molecule according to claim 8, wherein 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.

10. A modified PNA molecule according to any of claims 4 to 9, wherein the positively charged amino acids and amino acid analogs are selected from the group consisting of lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and ornithine (Orn).

11. A modified PNA molecule according to any of claims 4 to 10, wherein the non- charged amino acids and amino acid analogs are selected from Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin and the non-natural occurring amino acids 2-aminobutyric acid, β-cyclohexylalanine, 4-chlorophenylalanine, norleucine and phenylglycine.

12. A modified PNA molecule according to claims 4 to 11 , wherein the non-charged amino acids are selected from Ala, Val, Leu, lie, Pro, Phe, Trp, Met and the non- natural occurring non-polar amino acids β-cyclohexylalanine, 4-chlorophenylalanine and norleucine.

13. A modified oligonucleotide or PNA molecule according to any of the above claims, wherein the total number of amino acids in the Peptide is 15 or less, preferable 12 or less and more preferable 10 or less.

14. A modified oligonucleotide or PNA molecule according to any of the above claims, wherein the Peptide is selected from ( KFF ) ₃K (SEQ ID NO: 161) and subunits thereof comprising at least 3 amino acids, preferably ( KFF) ₃ (SEQ ID NO: 1).

15. A modified oligonucleotide or PNA molecule according to any of the above claims, wherein the Peptide is selected from FFRFFRFFR (SEQ ID NO: 6),

LLKLLKLLK (SEQ ID NO: 7), LLRLLRLLR (SEQ ID NO: 8), LLKKLAKAL (SEQ ID NO: 9), KFKVKFVVKK (SEQ ID NO: 11), LLKLLLKLLLK (SEQ ID NO: 12), LLKKLAKALK (SEQ ID NO: 13), RRLFPW WPFRRVC (SEQ ID NO: 14), GRR PW pwK PLic (SEQ ID NO: 15), LVKKVAT LKKI FSK KC (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.

16. A modified PNA molecule selected from the group consisting of:

H-KFFKFFKFFK-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 24), H-FFKFFKFFK-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 25), H-FKFFKFFK-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 26) , H-KFFKFFK-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 27) , H-FFKFFK-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 28), H-FKFFK-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 29) , H-KFFK-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 30), H-FFK-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 31) , H-FK-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 32), H-K-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 33) , H-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 34), H-KFFKFFKFF-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 35) , H-FFKFFKFF-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 36) , H-FKFFKFF-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 37) , H-KFFKFF-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 38) , H-FFKFF-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 39) , H-FKFF-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 40) , H-KFF-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 41) , H-FF-ado-CAT AGC TGT TTC-NH, (SEQ ID NO: 42) , H-F-ado-CAT AGC TGT TTC-NH₂ (SEQ ID NO: 43) , H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 18) , H-KFFKFFKFFK-ado-TGA CTA GAT GAG-NH₂ (SEQ ID NO: 44), H-KFFKFFKFFK-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 45), H-FFKFFKFFK-GGC-smcc-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 53) , H-FFRFFRFFR-GGC-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 54) , H-LLKLLKLLK-GGC-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 55), H-LLRLLRLLR-GGC-smcc-ado-TTC AAA CAT AGT-NH_: (SEQ ID NO: 56), H-LLKKLAKALK-GC-smcc-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 57), H-KRRWPWWP KK-C-smcc-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 58), H-KFKVKFVVKK-GC-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 59), H-LLKLLLKLLLK-C-smcc-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 60), H-FFKFFKFFK-GGC-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 61), H-KFFKFFKFFK-C-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 62), H-F-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 63), H-FF-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 64) , H-KFF-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 65) , H-FKFF-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 66), H-FFKFF-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 67) , H-KFFKFF-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 68) , H-FKFFKFF-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 69), H-FFKFFKFF-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 70), H-KFFKFFKFF-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 71), H-LLKKLAKALKG-a ex-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 21) , H-LLKKLAKALKG-ado-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 72) , H-KFFKFFKFFK-ado-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 73), H-KFFKFFKFFK-ahex-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 74), H₂N-KFFKFFKFFK-C-smcc-ado-CCA TCT AAT CCT-NH₂ (SEQ ID NO: 75) , H₂N-LLKKLAKALK-GC-smcc-ado-CCA TCT AAT CCT-NH (SEQ ID NO: 76) , H N-KFFKFF-C-smcc-ado-CCA TCT AAT CCT-NH, (SEQ ID NO: 77) , H-ado-TTC AAA CAT AGT-NH₂ (SEQ ID NO: 78), H₂N-KFFKVKFVVKK-C-smcc-ado-TTC AAA CAT AGT-NH, (SEQ ID NO: 79), H₂N-KFFKVKFVVKK-C-smcc-ado-TTG TGC CCC GTC-NH, (SEQ ID NO: 80), H₂N-KKFKVKFVVKKC-achc-β . ala-TTCAAACATAGT-NH;: (SEQ ID NO: 81), H-KFFKFFKFFK-achc-β . ala-TTCAAACATAGT-NH, (SEQ ID NO: 82), H₂N-KKFKVKFVVKKC-lcsmcc-ado-TTCAAACATAGT-NH_; (SEQ ID NO: 83), H₂N-KKFKVKFVVKKC-mbs-ado-TTCAAACATAGT-NH₂ (SEQ ID NO: 84), H₂N-KKFKVKFVVKKC-emcs-ado-TTCAAACATAGT-NH₂ (SEQ ID NO: 85), H₂N-KKFKVKFVVKKC-smph-ado-TTCAAACATAGT-NH, (SEQ ID NO: 86), H N-KKFKVKFVVKKC-amas-ado-TTCAAACATAGT-NH (SEQ ID NO: 87), H₂N-KKFKVKFVVKKC-smpb-ado-TTCAAACATAGT-NH, (SEQ ID NO: 88), H₂N-KKFKVKFVVKKC-lcsmcc-gly-TTCAAACATAGT-NH₂ (SEQ ID NO: 89), H₂N-KKFKVKFVVKKC-lcsmcc-β . ala-TTCAAACATAGT-NH, (SEQ ID NO: 90), H₂N-KKFKVKFVVKKC-lcsmcc-β . cypr-TTCAAACATAGT-NH₂ (SEQ ID NO: 91), H₂N-KKFKVKFVVKKC-lcsmcc-aha-TTCAAACATAGT-NH₂ (SEQ ID NO: 92), H₂N-KKFKVKFVVKKC-lcsmcc-adc-TTCAAACATAGT-NH₂ (SEQ ID NO: 93), H-KFFKFFKFFK-ado-ado-TTCAAACATAGT-NH₂ (SEQ ID NO: 94), H-KFFKFFKFFK-ado-Gly-TTCAAACATAGT-NH₂ (SEQ ID NO: 95), H-KFFKFFKFFK-ado-P-TTCAAACATAGT-NH₂ (SEQ ID NO: 96), H-KFFKFFKFFK-ado-aha-TTCAAACATAGT-NH, (SEQ ID NO: 97) , H-KFFKFFKFFK-ado-β . ala-TTCAAACATAGT-NH₂ (SEQ ID NO: 98) , H-KFFKFFKFFK-ado-achc-TTCAAACATAGT-NH₂ (SEQ ID NO: 99) , H-KFFKFFKFFK-Gly-ado-TTCAAACATAGT-NH₂ (SEQ ID NO: 100) , H-KFFKFFKFFK-Gly-Gly-TTCAAACATAGT-NH₂ (SEQ ID NO: 101), H-KFFKFFKFFK-Gly-P-TTCAAACATAGT-NH₂ (SEQ ID NO: 102) , H-KFFKFFKFFK-Gly-aha-TTCAAACATAGT-NH, (SEQ ID NO: 103) , H-KFFKFFKFFK-Gly-β . ala-TTCAAACATAGT-NH₂ (SEQ ID NO: 104) , H-KFFKFFKFFK-Gly-achc-TTCAAACATAGT-NH, (SEQ ID NO: 105) , H-KFFKFFKFFK-P-ado-TTCAAACATAGT-NH₂ (SEQ ID NO: 106), H-KFFKFFKFFK-P-Gly-TTCAAACATAGT-NH₂ (SEQ ID NO: 107), H-KFFKFFKFFK-P-P-TTCAAACATAGT-NH₂ (SEQ ID NO: 108), H-KFFKFFKFFK-P-aha-TTCAAACATAGT-NH, (SEQ ID NO: 109), H-KFFKFFKFFK-P-β.ala-TTCAAACATAGT-NH₂ (SEQ ID NO: 110), H-KFFKFFKFFK-P-achc-TTCAAACATAGT-NH₂ (SEQ ID NO: 111), H-KFFKFFKFFK-aha-ado-TTCAAACATAGT-NH, (SEQ ID NO: 112), H-KFFKFFKFFK-aha-Gly-TTCAAACATAGT-NH, (SEQ ID NO: 113), H-KFFKFFKFFK-aha-P-TTCAAACATAGT-NH₂ (SEQ ID NO: 114), H-KFFKFFKFFK-aha-aha-TTCAAACATAGT-NH, (SEQ ID NO: 115),

H-KFFKFFKFFK-aha-β. ala-TTCAAACATAGT-NH, (SEQ ID NO: 116), H-KFFKFFKFFK-aha-achc-TTCAAACATAGT-NH, (SEQ ID NO: 117), H-KFFKFFKFFK-β. ala-ado-TTCAAACATAGT-NH, (SEQ ID NO: 118), H-KFFKFFKFFK-β. ala-Gly-TTCAAACATAGT-NH₂ (SEQ ID NO: 119), H-KFFKFFKFFK-β. ala-P-TTCAAACATAGT-NH, (SEQ ID NO: 120),

H-KFFKFFKFFK-β. ala-aha-TTCAAACATAGT-NH₂ (SEQ ID NO: 121), H-KFFKFFKFFK-β. ala-β. ala-TTCAAACATAGT-NH, (SEQ ID NO: 122), H-KFFKFFKFFK-β. ala-achc-TTCAAACATAGT-NH, (SEQ ID NO: 123), H-KFFKFFKFFK-P-p-TTCAAACATAGT-NH₂ (SEQ ID NO: 124), H-KFFKFFKFFK-P-P-TTCAAACATAGT-NH, (SEQ ID NO: 125), H-KFFKFFKFFK-K-K-TTCAAACATAGT-NH: (SEQ ID NO: 126), H-KFFKFFKFFK-F-F-TTCAAACATAGT-NH, (SEQ ID NO: 127), H-KFFKFFKFFK-F-K-TTCAAACATAGT-NH, (SEQ ID NO: 128), H-KFFKFFKFFK-K-F-TTCAAACATAGT-NH, (SEQ ID NO: 129), H-KFFKFFKFFK-phg-ado-TTCAAACATAGT-NH, (SEQ ID NO: 130), H-KFFKFFKFFK-phg-Gly-TTCAAACATAGT-NH, (SEQ ID NO: 131), H-KFFKFFKFFK-phg-P-TTCAAACATAGT-NH, (SEQ ID NO: 132), H-KFFKFFKFFK-phg-aha-TTCAAACATAGT-NH₂ (SEQ ID NO: 133), H-KFFKFFKFFK-phg-β.ala-TTCAAACATAGT-NH₂ (SEQ ID NO: 134), H-KFFKFFKFFK-phg-achc-TTCAAACATAGT-NH, (SEQ ID NO: 135), H-KFFKFFKFFK-achc-ado-TTCAAACATAGT-NH, (SEQ ID NO: 136), H-KFFKFFKFFK-achc-Gly-TTCAAACATAGT-NH, (SEQ ID NO: 137), H-KFFKFFKFFK-achc-P-TTCAAACATAGT-NH, (SEQ ID NO: 138), H-KFFKFFKFFK-achc-aha-TTCAAACATAGT-NH, (SEQ ID NO: 139), H-KFFKFFKFFK-achc-β.ala-TTCAAACATAGT-NH₂ (SEQ ID NO: 140) or H-KFFKFFKFFK-achc-achc-ττcAAACATAGT-NH₂ (SEQ ID NO: 141) wherein the linking groups are as defined in claim 5.

17. A modified PNA molecule according to any of the claims 3 to 16, wherein the PNA sequence is complementary to at least one nucleotide sequence in a bacteria.

18. A modified PNA molecule according to claim 17 wherein said nucleotide sequence is a ribosomal RNA, messenger RNA or DNA sequence.

19. A modified PNA molecule according to any of claims 3 to 18, wherein the PNA sequence is in a parallel or anti-parallel orientation.

20. A modified PNA molecule according to any of the claims 17 to 19, wherein the functioning of the said nucleotide sequence is essential for the growth or survival of the bacteria and said functioning is blocked by the PNA sequence.

21. A modified PNA molecule or a modified oligonucleotide according to any of the claims 1 to 20 for uses in the treatment of infectious diseases or in disinfection of non-living objects.

22. Use of a modified PNA molecule or a modified oligonucleotide according to any of claims 1 to 20 in the manufacture of a medicament for the treatment of infectious diseases.

23. Use of a modified PNA molecule or a modified oligonucleotide according to any of claims 1 to 20 in the manufacture of a composition for the treatment or prevention of bacterial infections.

24. A composition for use in the treatment or prevention of bacterial growth or survival, comprising a modified PNA molecule or a modified oligonucleotide according to any of claims 1 to 20.

25. A composition according to claim 24 further comprising an antibiotic.

26. A composition according to claim 24 or 25 comprising two or more modified PNA molecules according to claims 3 to 20.

27. A method of treating an infectious disease, comprising administering to a patient in need thereof an efficient amount of a modified PNA molecule or a modified oligonucleotide according to claims 1 to 20 or a composition according to any of claims 24 to26.

28. A method of disinfecting non-living objects, comprising administering to said non-living object an efficient amount of one or more modified PNA molecules or a modified oligonucleotide according to claims 1 to 20 or a composition according to any of claims 24 to26.

29. A method according to claim 28 where said object is selected from surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tools, dairy inventory and tools, etc.

30. Use of a modified PNA molecule according to any of claims 3 to 20 in the identification of PNA sequences which are effective in blocking essential functions in bacteria, wherein different PNA sequences are incorporated in the modified PNA molecule and tested for their ability to inhibit or reduce the growth of the bacteria.

31. Method of identifying a PNA sequence, which is useful in inhibiting or reducing the growth of one or more bacteria, comprising mixing modified PNA molecules according to any of claims 3 to 20, comprising different PNA sequences, with one or more selected bacteria, the PNA sequences being selected so as to be complementary to at least one nucleotide sequences for each selected bacteria, and identifying the PNA sequences which are effective in inhibiting or reducing the growth said one or more bacteria.

32. A molecule according to claims 4 to 20, wherein the PNA sequence comprises 5-20 nucleobases, in particular 7-15 nucleobases and most particular 8-12 nucleobases.

33 . A modified PNA molecule selected from the group consisting of: H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCCCTCTC-Lys-NH₂ (SEQ ID NO: 22),

H-KFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH₂ (SEQ ID NO: 46), H-FKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID NO: 47), H-FFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID NO: 48),

H-KFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID NO: 49),

H-FKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID NO: 50), H-FFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID NO: 51) or H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH, (SEQ ID

NO: 52) wherein the linking groups are as defined in claim 5.