WO2004001055A2

WO2004001055A2 - Pna prodrugs

Info

Publication number: WO2004001055A2
Application number: PCT/DK2003/000416
Authority: WO
Inventors: Nadia Bendifallah; Peter Eigil Nielsen
Original assignee: Santaris Pharma AS
Current assignee: Roche Innovation Center Copenhagen AS
Priority date: 2002-06-20
Filing date: 2003-06-20
Publication date: 2003-12-31
Anticipated expiration: 2004-12-20
Also published as: AU2003240435A8; AU2003240435A1; WO2004001055A3

Abstract

The present invention concerns novel drugs for use in combating various diseases. More particular, the invention concerns peptide nucleic acid (PNA) prodrugs and methods for their preparation and pharmaceutical use. The PNA prodrugs comprise a suitable carrier attached to the PNA by a cleavable linker, whereby the PNA prodrug exhibit enhanced transport across cell membranes with subsequent intracellular release of the PNA, improving the access of the PNA to potential cellular/nuclear targets.

Description

PNA PRODRUGS

The present invention concerns novel drugs for use in combating various diseases. More particular, the invention concerns peptide nucleic acid (PNA) prodrugs and methods for their preparation.

BACKGROUND OF THE INVENTION

Antisense agents offer a novel strategy in combating diseases, as well as opportunities to employ new chemical classes in the drug design.

Oligonucleotides can interact with native DNA and RNA in several ways. One of these is duplex formation between an oligonucleotide and a single stranded nucleic acid. Another is 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 has wide applications.

Peptide nucleic acids (PNAs) 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 In PNA, the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 (1 )).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 (2)). The pseudo-peptide backbone provides superior hybridization properties (Egholm et al. 1993 (2)), resistance to enzymatic degradation (Demidov et al. 1994 (3)) and access to a variety of chemical modifications (Nielsen and Haaima 1997 (4), WO 94/25477, WO 98/03542).

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 the melting point temperature (Tm). 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 melting point temperature.

Furthermore, homopyrimidine PNA oligomers form extremely stable PNA₂- DNA (RNA) triplexes with sequence complementary targets in DNA or RNA oligomers. Finally, PNAs 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, PNAs are resistant to degradation by enzymes unlike nucleic acids and peptides.

PNAs can be used as a tool in molecular biology and biotechnology, for diagnostic purposes and development, but it is also a lead compound for the development of gene-targeted drugs applying antigene or antisense strategy.

For antigene or antisense application, target bound PNAs can cause steric hindrance of DNA and RNA polymerases, reverse transcription, telomerase and of the ribosomes (Hanvey et al. 1992 (5), Knudsen et a. 1996 (6), Good and Nielsen 1998 (7,8)) by targeting among others DNA, mRNA, rRNA, or tRNA. The sequence-specific inhibition by PNAs could potentially be exploited for therapeutic applications. However, cellular delivery of the PNA is required for antigene and antisense applications. Previous work has shown that cells only poorly take up naked PNA. Within the last couple of years a variety of methods have been devised to address this problem.

WO 99/05302 discloses a PNA conjugate consisting of PNA and the carrier peptide transportan, which peptide may be used for transport across a lipid membrane for delivery of the PNA to intracellular targets.

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.

WO 96/11205 discloses PNA conjugates, wherein a conjugated moiety may be placed on terminal or non-terminal parts of the backbone of PNA in order to functionalise the PNA. The conjugated moieties may be reporter enzymes or molecules, steroids, carbohydrate, terpenes, peptides, proteins, etc. It is suggested that the conjugates among other properties may possess improved transfer properties for crossing cellular membranes.

WO 98/52614 discloses a method of enhancing transport over biological membranes. 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 or amidino sidechain moiety and wherein at least 6 contiguous subunits contain guanidino and/or amidino sidechains. A preferred transporter polymer is a polypeptide containing 9 arginine ((Arg)₉).

Work carried out by Ljungstrøm et al in 1999 (9) showed that endosomal cellular uptake of PNAs is greatly increased, when fatty acids such as adamantyl acetic acid is coupled to the terminal amine of the PNA. However PNA-fatty acid conjugates have limited solubility in an aqueous environment and become easily entrapped in lipid compartments inside the cell, such as various membranes, which could limit their access to the potential targets such as mRNA, tRNA, sRNA or DNA.

In order to increase the efficacy of PNA drugs, when combating diseases, there is a need of releasing the PNA from its carrier at the target site.

An oligonucleotide prodrug reactive with a cellular or tissue enzyme, which cleaves the lipophilic group from the derivatised nucleotide, thereby regenerating the parent oligonucleotide is disclosed in WO96/07392. The oligonucleotide prodrug comprises at least six covalently linked nucleotides, at least one of which is derivatised with a lipophilic chemical group containing an ester or amide covalently attached to the nucleotide at a 5' phosphate, 3' phosphate, or an intemucleotidic phosphate linkage.

Comprising a charge neutral backbone compared to oligonucleotides, PNA prodrugs need only to possess the effect of promoting the transport of PNAs across membranes, with subsequent cleaving off the carrier from the PNA antisense-targeting group, at the target site.

Work presented by Pooga et al (17) in 1998 showed that PNA oligomers linked to cell penetrating peptides by disulfide bonds dissociated in intracellular milieu, permitting the PNA to associate with target mRNA in the cytosol. These results indicate that PNA constructs with carrier peptides are promising candidates for antisense therapeutics. However, disulphide bonds are unstable and may dissociate from the PNA in pharmaceutical compositions before penetration has taken place.

SUMMARY OF THE INVENTION

The present invention relates to novel Peptide Nucleic Acid (PNA) prodrugs comprising a suitable carrier attached to the PNA through an in vitro stable, enzymatically cleavable moiety, allowing cellular delivery of PNA prodrugs. The PNA prodrugs exhibit enhanced transport across cell membranes with subsequent intracellular liberation of the PNA by intracellular processes, thereby improving the access of the PNA to potential targets such as mRNA, rRNA, tRNA or DNA.

The uptake of PNA prodrugs of the present invention achieves a different intracellular distribution as compared to non-cleavable PNA drugs, as the released PNA appear to be nuclear associated although without full nuclear localisation. Such a nuclear localisation is advantageous for antigene- targeting and antisense-targeting of exon-intron splice junctions.

Accordingly, the present invention relates to a modified PNA prodrug of formula (I)

Q - L1-E-L2 - PNA (I)

wherein Q is a carrier moiety; E is a cleavable moiety; Li and L₂ are, independently, a linker or a bond; and PNA is a peptide nucleic acid oligomer.

In a preferred embodiment of the invention, E is an amino acid of the formula (II)

- HN - A- B - CO - (II)

in which - A - B - is a group of formula (III)

(III) in which X and Y, independently, are preferably an alkylene group, a C₂-n- alkenylene, a C∑ n-alkynylene, a phenylene group, or a benzylidene group. The ester bond is sensitive to cleavage by an enzyme.

In yet another preferred embodiment, the - A - B -- is a group of formula (IV)

(IV)

In a preferred embodiment of the invention, the carrier moiety of the PNA prodrug contains a lipid, a cell penetrating peptide such as Penetratin/Antp (Brugidou et al 1995 (10, 11)), Tat (Falnes et al 2001 (12)), NLS (Zanta et al 1999 (13)), Transportan (Lindgren et al 2000 (14), Magzoub et al 2001 (15), Pooga et al 1998 (16)), (Arg)_n= -ι₅, a peptidomimetic, or a hormone. The carrier moiety is linked to the PNA sequence via the amino (N-terminal) or carboxy (C-terminal) end.

In a preferred embodiment, the PNA prodrug of the invention is prepared from an amino acid of the formula (V)

R¹ - HN - A - B - CO - R² (V)

in which the A - B bond is sensitive to cleavage by an enzyme, R¹ is H, Boc, Fmoc or other suitable solid phase protecting groups, R² is -OH, -OR³, -SR³, -OCOR³,

R³ is an alkyl, a C₂-_n-alkenyl, a C₂-_n-alkynyl, an aryl, or an alkylaryl, R⁴ is H, F, Cl, or Br and R⁵ is H, or -SO₃H.

In yet another preferred embodiment, a method of synthesising a PNA prodrug of the invention comprising cleavable moieties by use of solid-phase synthesis is claimed.

Medical applications of the compounds of the present invention include treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders. PNA prodrugs that bind to targets in single stranded and double stranded DNA as well as to mRNA, rRNA and tRNA by a variety of mechanisms may be developed into antigene or antisense drugs by targeting specific sequences of specific genes. In this way the expression of the targeted gene can be inhibited, or in desired cases activated, and the level of a disease related gene product thereby regulated.

DEFINITIONS

An "alkyl group" according to the present invention refers to a Cι-_n-alkyl, wherein n' can be from 2 through 15, representing a branched or straight alkyl group having from one to the specified number of carbon atoms. Typical C-ι-₆-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso- propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl saturated as well as non-saturated groups.

The terms "C₂-_n-alkenyl" wherein n' can be from 3 through 15, as used herein, represents an olefinically unsaturated branched or straight group having from 2 to the specified number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, allyl, iso-prophenyl, 1 ,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like. The terms "C₂._n'-alkynyl" wherein n' can be from 3 through 15, as used herein, represent an unsaturated branched or straight group having from 2 to the specified number of carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl and the like.

The term "aryl group" as used herein refers to an aryl including aromatic rings, such as carboxylic aromatic rings selected from the group consisting of phenyl, naphthyl, (1-naphtyl or 2-naphtyl) optionally substituted with halogen, amino, hydroxy, Ci-β-alkyl or C-ι-₆-alkoxy.

An "alkylaryl group" according to the present invention refers to a straight or branched saturated carbon chain containing from 1 to 6 carbons substituted with an aromatic carbohydride; such as benzyl, phenethyl, 3-phenylpropyl, 1- naphtylmethyl, 2-(1-naphtyl)ethyl and the like.

The term a charge neutral oligomer of the present invention refers to the charge neutral backbone of the PNA oligomer, comprising N-(2- aminoethyl)glycine.

DETAILED DESCRIPTION OF THE INVENTION

Figure 1 : IMR-92 cellular uptake of the synthesized PNA-3 drug (non- cleavabgle) and PNA-5 prodrug (cleavable) compared to the control.

In accordance with the present invention, PNA prodrugs exhibiting enhanced cellular uptake and distribution are provided. The PNA prodrugs are assembled from a suitable carrier attached to the PNA by a among others, a cleavable moiety, whereby the carrier is cleaved off from the PNA following transport across cell membranes, thereby preventing intracellular entrapment of the PNA in lipid or other compartments inside the cell. The PNA prodrugs of the present invention have the formula (I), wherein E is the cleavable moiety. In another preferred embodiment of the present invention, the cleavable moiety comprises an ester.

The PNA prodrug of the present invention may be used in other PNA conjugates such as defined in the PCT Publication WO 01/27261 enhancing the cellular distribution and nuclear association of the PNA.

Solid Phase Synthesis of PNA Prodrugs

The principle of anchoring molecules during a reaction onto a solid matrix is known as Solid Phase Synthesis (Merrifield, B 1986 (18)). Multiple methods for the stepwise solid phase assembly of monomers into peptides are established. PNA prodrugs of the present invention are prepared as outlined in WO98/53801.

In a preferred embodiment of the invention, a method of synthesising PNA prodrugs of the invention comprising cleavable moieties, by use of solid- phase synthesis, is claimed. PNA oligome sation (and especially cleavage from the resin) involves rather harsh acid and/or alkaline treatment. Surprisingly, it has been found that the cleavable moieties are easily incorporated into PNA prodrugs using solid phase polymerisation, deprotection and cleavage conditions. However, a necessary precaution to be taken is the employment of carefully dried ether for precipitation of the product after cleavage from the resin, in order to avoid hydrolysis of the cleavable moieties.

Fragment Coupling of PNA Prodrugs

Fragment coupling is an alternative method of producing compounds of the invention. For instance, following independent solid phase synthesis of the carrier moiety containing an -SH function (e.g. a cystein) respectively the PNA containing the cleavable linker, the carrier moiety may be fragment- coupled to the cleavable linker by applying e.g. succinimidyl 4-[N- maleimidomethyl]-cyclohexan-1-carboxylat (SMCC). Other coupling chemistries known to the skilled artisan may also be applied (Wong 1993 (23)).

Pharmaceutical Compositions

The PNA prodrugs of the present invention are used in the manufacture of medicaments for the treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or for disinfecting non-living objects, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools and the like.

Within the present invention, the compounds of the invention may be prepared in the form of pharmaceutically acceptable salts, especially acid- addition salts, including salts of organic 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, Berge et al 1977 (19), 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 prodrugs 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 one aspect, the invention concerns the manufacture of a composition for treating or preventing bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or disinfecting non-living objects, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools and the like.

Typical compositions include a compound of the invention 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 an 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, alcohol's, polyethylene glycol's, polyhydroxyethoxylated castor oil, peanut oil, olive oil, glycine, gelatin, lactose, terra alba, sucrose, glucose, cyclodextrine, 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 flavoring 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 coloring substances and the like, which do not deleteriously react with the active compounds.

For therapeutic or prophylactic treatment, the PNA prodrug of the invention can be formulated in a pharmaceutical composition, which may include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anaesthetics, and the like in addition to PNA.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavourings, diluents, emulsifiers, dispersing aids or binders may be desirable.

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.

Formulations for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives.

For nasal administration, the preparation may contain a compound of the invention dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilising agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrine, 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, cornstarch, and/or potato starch. A syrup or elixir can be used in cases where a sweetened vehicle can be employed.

In yet another aspect, the invention concerns the treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or treatment of non-living objects.

Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.

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 the invention admixed with a pharmaceutically acceptable carrier or diluent.

Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilises DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated. Further, since each cell of multicellular eukaryotes can be treated since they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Furthermore, many of the organelles, (e.g. mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic PNA prodrug. As used herein, therapeutics is meant to include the eradication of a disease state, by killing an organism or by control of erratic or harmful cellular growth or expression. EXAMPLES

General

The reagents used were Phtalid (Aldrich), 1 ,3-dicyclohexylcarbodiimide (DCC, Aldrich), N-t-Boc-Glycine (Sigma), 4-dimethylaminopyridine (DMAP, Aldrich), 1-pyrenebutyhc acid (Aldrich), Decanoic acid (Deca, Aldrich), 1- Adamantaneacetic acid (Ada, Aldrich). TLC was performed on silica 60 (Merck 5554 aluminium sheet), dichloromethane (LAB-SCAN), and diethyl ether (LAB-SCAN). The two last mentioned were dried over molecular sieves before use.

¹H and ¹³C-NMR spectra were obtained at either 250 MHz (Bruker AMX 250) or at 400 MHz (Varian Unity 400) in 5 mm tubes.

FAB mass spectra were recorded on a Jeol Hx 110/110 mass spectrometer. MALDI-TOF mass spectra recorded on a Kratos Compact MALDI II instrument operating in the positive ion mode using 3.5-dimethoxy-4- hydroxycinnamic acid as the matrix.

Abbreviations

Ada: 1 -adamantaneacetic acid

Ado: 8-amino-3,6-dioxa-octanoic acid (alias eg-i)

Boc: tert-Butyloxycarbonyl

BMB2: 2-([N-Boc-glycyl]oxymethyl)benzoic acid

DCC: 1 ,3-Dicyclohexylcarbodiimide

DCU : N,N'-Dicyclohexylurea

Deca: Decanoic acid

DIEA: Diisopropylethylamine

DMAP: 4-Dimethylaminopyridine

DMEM: Dulbecco's Modified Eagle's Medium

DMSO: Dimethyl sulfoxide FI Lys- Fluorescein Lysin monomer (6-0-[N-5-Boc-amino-5- carboxypentyl)carbamoylmethyl]fluorescein ethyl ester)

HPLC: High performance liquid chromatography

MBHA: 4-(Methylbenzhydryl)-amine

NMP: N-Methylpyrrolidone

PBS: Phosphate-buffered saline

PI: Propidium iodide

Pyr: 1-Pyrenebutyric acid

SMCC: Succinimidyl 4-[N-maleimidomethyl]-cyclohexan-1 - carboxylate

TFA: Trifluoric acid

TJ: Pseudoisocytosine

EXAMPLE 1

Synthesis of the cleavable monomer

2-hydroxymethylbenzoic acid

Phtalide (5.0 g, 37.28 mmol) was dissolved in methanol (20 mL) and water (20 mL). NaOH (10 M, 8 ml) was added, and the mixture was refluxed for 90 min. The reaction mixture was then cooled to room temperature, and the free acid was precipitated with 4 M HCI (ca. 10 ml). The resulting precipitate was filtered off, washed with ice-cold water and finally dried in vacuo to give a white crystalline product (5.03 g, 89 %). Melting point (M.p.): 101-105 °C (litt. 120.5-121.5 °C). ¹H-NMR (DMSO-d₆) δ 8.12 (dd, 1 H), 7.61 (m, 1 H), 7.47 (m, 2H), 4.86 (s, 2H), ¹³C-NMR (DMSO-d₆) δ 133.79, 132.01 , 130.32, 128.00, 64.61 (it was not possible to see the C=0 signal due to the low sample concentration). FAB⁺MS: 153.12 (M+ H⁺). EXAMPLE 2

2-([N-Boc-glvcvn-oxymethyl) benzoic acid (BMB2)

A symmetrical anhydride was formed from N-Boc-glycine (7.72 g, 44.06 mmol) and DCC (4.55 g, 22.03 mmol) in dry dichloromethane (25 ml). The mixture was stirred at 0°C for 30 min. The resulting N,N'-dicyclohexylurea (DCU) was filtered off and the filtrate was added directly to a suspension of 2-hydroxymethylbenzoic acid (Gilman et al 1940 (20)) in dry dichloromethane (25 ml). DMAP (0.19 g, 1.54 mmol) was added and the mixture was refluxed overnight. The reaction mixture was washed with saturated aqueous NaHC0₃ (3 x 50 ml). The combined aqueous extracts were acidified with 4 M HCI to pH 2 and the resulting precipitate was filtered off, washed with ice-cold water and finally dried in vacuo to give a white crystalline product (2.98 g, 63%) (M.p.: 122-124°C). ¹H-NMR (DMSO-d₆), δ 7.93 (d, 1 H, J= 7.5 Hz), 7.55 (m, 2H), 7.44 (m, arom), 7.28 (t, 1 H, J= 6.1 Hz), 5.49 (s, 2H), 3.79 (d, 2H, J= 6.1 Hz), 1.38 (s, 9H). ¹³C-NMR (DMSO-d₆) δ 170.33, 168.0, 155.99, 137.38, 132.21 , 130.60, 129.28, 127.83, 127.53, 78.38, 64.18, 42.12, 28.24. FAB⁺HRMS: 310.1265 (M+H⁺, calc. for C₁₅Hι₉NO₆+H⁺ 310.1291).

Reaction scheme of2-([N-Boc-glycyl]oxymethyl)benzoic acid (BMB2).

(a) NaOH, MeOH aq., Δ (b) 4N HCI (89%) (c) (Boc-Glyj_∑-O/DMAP/DCM, Δ (d) 4N HCI (63%). Synthesis of the PNA Prodrugs and PNA Drugs

PNA-1 : Pyr-BMB2-TJ-ado-ado-ado-CTTTCTT-NH₂ PNA-2: Deca-ado-FI_Ly_S-ado-CATAGTATAAGT-LysNH₂ PNA-3: Ada-ado-FI_Lys-ado-CATAGTATAAGT-LysNH₂

PNA-4: Deca-BMB2- ado-FI_Lys-ado-CATAGTATAAGT-LysNH₂ PNA-5: Ada- BMB2-ado-FI_Ly_S-ado-CATAGTATAAGT-LysNH₂

EXAMPLE 3

PNA-1 : Pyr-BMB2-TJ-ado-ado-ado-CTTTCTT-NHg

The PNA prodrug was synthesised on a Boc-T-4-methylbenzhydrylamine resin (loading 0.12 mmol/g) using the standard synthetic protocol. For the incorporation of 1-pyrenebutyric acid and 2-([N-Boc-glycyl]oxymethyl) benzoic acid, the coupling was allowed to proceed for 60 min.

The resulting PNA prodrug was deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/trifluoromethane sulfonic acid/TFA (1/1/2/6, v/v) and the crude material was precipitated and washed with anhydrous diethylether. The PNA prodrugs were then purified by reversed-phase high performance liquid chromatography (HPLC) and characterised by MALDI-TOF mass spectroscopy.

The synthesised PNA-1 prodrug: Pyr-BMB2-TJ-ado-ado-ado-CTTTCTT-NH₂. EXAMPLE 4

PNA-2: Deca-ado-F _ys-ado-CATAGTATAAGT-LvsNH?

The PNA drug was synthesised on a Boc-L-Lys(2-chlorobenzyloxycarbonyl)- 4-methylbenzhydrylamine resin (loading 0.15 mmol/g) using the standard synthetic protocol (Christensen et al 1995 (21 )). For the incorporation of decanoic acid and the fluorescein-lysin monomer (6-O-[N-(5-Boc-amino-5- carboxypentyl)carbamoylmethyl]fluorescein ethyl ester) (Lohse et al 1997 (22)), the coupling was allowed to proceed for 60 min.

The resulting PNA drug was deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/thfluoromethane sulfonic acid/TFA (1/1/2/6, v/v) and the crude material was precipitated and washed with anhydrous diethylether. The PNA drug was then purified by reversed-phase HPLC and characterised by MALDI-TOF mass spectroscopy.

EXAMPLE 5

PNA-3: Ada-ado-Fli^-ado-CATAGTATAAGT-LvsNH?

The PNA drug was synthesised on a Boc-L-Lys(2-chlorobenzyloxycarbonyl)- 4-methylbenzhydrylamine resin (loading 0.15 mmol/g) using the standard synthetic protocol (Christensen et al 1995 (21)). For the incorporation of 1- adamantaneacetic acid, acid and the fluorescein-lysin monomer (6-O-[N-(5- Boc-amino-5-carboxypentyl)carbamoylmethyl]fluorescein ethyl ester) (Lohse et al 1997 (22)), the coupling was allowed to proceed for 60 min.

The resulting PNA drug was deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/trifluoromethane sulfonic acid/TFA (1/1/2/6, v/v) and the crude material was precipitated and washed with anhydrous diethylether. The PNA drug was then purified by reversed-phase HPLC and characterised by MALDI-TOF mass spectroscopy. EXAMPLE 6

PNA-4: Deca-BMB2- ado-Flι _ys-ado-CATAGTATAAGT-LvsNH?

The PNA prodrug was synthesised on a Boc-L-Lys(2- chlorobenzyloxycarbonyl)-4-methylbenzhydrylamine resin (loading 0.15 mmol/g) using the standard synthetic protocol (Christensen et al 1995 (21)). For the incorporation of decanoic acid, 2-([N-Boc-glycyl]oxymethyl)benzoic acid and the fluorescein-lysin monomer (6-O-[N-(5-Boc-amino-5- carboxypentyl)carbamoylmethyl]fluorescein ethyl ester) (Lohse et al 1997 (22)), the coupling was allowed to proceed for 60 min.

The resulting PNA prodrug was deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/trifluoromethane sulfonic acid/TFA (1/1/2/6, v/v) and the crude material was precipitated and washed with anhydrous diethylether. The PNA prodrug was then purified by reversed- phase HPLC and characterised by MALDI-TOF mass spectroscopy.

EXAMPLE 7

PNA-5: Ada-BMB2-ado-Flι _y ado-CATAGTATAAGT-LvsNH?

The PNA prodrug was synthesised on a Boc-L-Lys(2- chlorobenzyloxycarbonyl)-4-methylbenzhydrylamine resin (loading 0.15 mmol/g) using the standard synthetic protocol (Christensen et al 1995 (21)). For the incorporation of 1 -adamantaneacetic acid, 2-([N-Boc- glycyl]oxymethyl)benzoic acid and the fluorescein-lysin monomer (6-O-[N-(5- Boc-amino-5-carboxypentyl)carbamoylmethyl]fluorescein ethyl ester) (Lohse et al 1997 (22)), the coupling was allowed to proceed for 60 min.

- ado - CATAGTATAAGT - LysNH

The synthesised PNAS prodrug: Ada-BMB2-ado-FI_LYS-CATAGTATAAGT- LysNH₂.

EXAMPLE 8

Solid Phase Synthesis procedure

The PNA prodrugs, respective PNA drugs were assembled using a conventional solid phase synthesis on a preloaded 4-(methylbenzhydryl)- amine (MBHA).

The synthesis cycle was as follows:

I. Boc deprotection by 5% m-cresol in TFA (2 x 4 min.)

II. Wash

III. Coupling (20 min)

IV. Wash V. Capping (Ac₂O/Collidin/DMF (1 x 4 min.)

VI. Wash

The Kaiser test was applied to determine the termination of the coupling process.

Incorporation of the cleavable moiety into PNA oligomers proceeded under standard PNA oligomerisation conditions and yielded products that were readily purified by reversed phase HPLC. The only necessary precaution taken was the employment of carefully dried ether for precipitation of the product after cleavage from the resin in order to avoid hydrolysis of the cleavable moiety.

EXAMPLE 9

Fragment Coupling: an Alternative Way of Preparing PNA Prodrugs and PNA Drugs

Fragment coupling is an alternative method of producing compounds of the invention. Following independent solid phase synthesis of the carrier moiety containing an -SH function (e.g. a cystein) respectively of the PNA containing the cleavable linker, the carrier moiety may be fragment coupled to the cleavable linker by applying succinimidyl 4-[N-maleimidomethyf]- cyclohexan-1-carboxylat (SMCC). Other coupling chemistries known to the skilled artisan may also be applied (Wong 1993 (23)).

PNA fragments were diluted in NMP:DMSO (8:2) for 45 min. SMCC diluted in NMP was added with DIEA and the mixture was allowed to react for three hours while stirred. The formed PNA-maleimide conjugate was washed three times with diethylether.NMP (9:1 ) to remove excessive SMCC and subsequently three times with diethylether before dried with nitrogen. The carrier was diluted in 0.1 M NH₄CO₃. DTT was added and the mixture was allowed to react for three hours in nitrogen before pH was adjusted to 7.0 by addition of acetic acid. The formed carrier conjugate was separated on a sephadex G-25 gel before mixed with the previous formed PNA-SMCC derivate diluted in DMF. The reaction was stirred over night and the formed PNA prodrug or PNA drug was collected the following day. In vitro analysis

EXAMPLE 10

Tissue Extracts and Stability Analysis

The HPLC system (Waters) consisted of an Alliance 2690 (pump, autosampler and degasser), a PDA UV absorbance detector Model 996 (195 nm - 600 nm) and Millennium³² Chromatography software version 3.2. HPLC separation was performed on a Waters Symmetry 300™ C18, 2.1 x 150 mm (3.5 μm particles with 300 A pore size) analytical column (Waters) equipped with a Zorbax Eclipse XDB-C18 (5 μm particles with 80 A pore size) guard column (Agilent) using linear gradient elusion of solvent A (0.1 % TFA in water) and solvent B (0.1% TFA in acetonitrile) from 2% to 75% solvent B over 8 minutes. The column was operated at 50°C. Samples were kept in the autosampler at 5°C. Solvent flow was 0.4 ml/min. Polypropylene (PP) test tubes and containers (Sarsted) were generally used for all solutions containing the test PNA. Samples were centrifuged in a Hettich Rotina 46R centrifuge and evaporated to dryness in a SpeedVac AES 2010 from Savant. NMRI mice (female, 25 g bodyweight) were obtained from M&B (Denmark) for in vivo studies. Plasma and tissue from sacrificed animals were used for in vitro stability/metabolism studies.

EXAMPLE 11

Preparation of Tissue Homoqenates for in vitro Studies

The animal was sacrificed and the relevant tissues (liver, kidneys) rapidly excised. The tissue was immediately placed in 0.25 M sucrose at 0°C for rapid cooling and removal of external blood. After cooling (3-5 min) the tissue was dried by blotting with paper, and subsequently weighed and transferred to clean PP test tubes. To each tissue was added 0.25 M sucrose in water to a final concentration of 150 mg tissue/ml, using a density of 1 mg/ml for the tissue. The tissue was cut into pieces and homogenised for 1-5 min (depending on tissue) in an Ultra-Turrax® T25 homogeniser (IKA) followed by centrifugation of the homogenate in a refrigerated centrifuge (4°C) for 30 minutes at 3000 rpm (corresponding to approx. 1000 x g) to isolate sub- cellular fractions. The supernatant was carefully decanted (~ the post- mitochondrial supernatant), transferred to polypropylene containers, and stored at minus 80°C pending use.

EXAMPLE 12

In vitro Metabolism Studies

A mixture of 0.025 ml in 0.1 M Tris buffer pH 7.4, 0.135 ml of water and 0.025 ml of the tissue homogenate (or plasma) was pre-incubated at 37°C for 2 minutes, before addition of 0.015 ml PNA stock solution (1 mg/ml). After incubation for the desired time period, the enzymatic reactions were stopped by adding 0.300 ml of 16.6% acetonitrile in 0.1% TFA in water, and the sample immediately transferred to an ice-water bath (0°C). The mixture was frozen at minus 18°C for 30 minutes. For analysis, the homogenate mixtures were thawed at 4°C and centrifuged at 3000 rpm for 10 min. (approx. 1000 x g) at 4°C. The supernatant (0.200 ml) was transferred directly to the HPLC autosampler vials and 0.010 ml aliquots were injected into the HPLC system. Blind samples were prepared by replacing the PNA test compound with water. The blind samples were incubated and analysed as described for the test samples. Recovery in the incubated samples was calculated from the HPLC area using a reference solution.

The stability of the PNA-1 prodrug in undiluted tissue homogenates (rat plasma, rat kidney and rat liver) compared to control (Tris buffer, pH 7.4 at 37°C) was estimated to be up to 2000-3000 times less stable (Table 1). However, the PNA-1 prodrug showed a somewhat limited stability in the buffer with a ty₂ of 6 hours.

Table 1 Stability analysis of PNA-1 prodrug in rat tissue homogenates.

EXAMPLE 13

In vivo analysis

Cellular uptake

The IMR-90 cell line (human foetal fibroblast) was obtained from American Type Culture Collection and grown in Dulbecco^'s modified Eagle^'s medium (DMEM) supplemented with penicillin/streptomycin (10 μg/mL) (antibiotics) and 10% foetal calf serum (Gibco, Invitrogen, Denmark). Cell cultures was maintained at 37°C in a humidified atmosphere containing 5% CO₂ (standard conditions).

For uptake analysis of the PNA prodrug, respective PNA drug, the cells were cultivated on 4 wells chamber slides (Nunc, Invitrogen, Denmark). The cells were seeded in a concentration of 20 x 10³/ml. The following day, the semi confluent cells were prepared for transfection by washing in fresh DMEM containing antibiotics and 2% foetal calf serum. The cells were subsequently added fresh DMEM containing antibiotics and 2% foetal calf serum and incubated 40 minutes at the standard conditions, before replacement of the media with the transfection mixture. The transfection mixture was prepared by diluting 4 μl of lipofectAMINE (2 mg/ml) (Life Technologies) in 46 μl DMEM (without serum) and, in a separate tube, diluting PNA in 50 μl DMEM (without serum) to a final concentration of 4 μM (approximately 18 μg/ml). The lipofectAMINE dilution and the PNA dilution was then gently mixed and left for equilibration at room temperature for 30 minutes. 400 μl DMEM containing antibiotics and 2% foetal calf serum was added to the combined PNA/lipofectAMINE solution and the resulting transfection mixture was gently layered upon the cells. After 14-16 hours of incubation, at standard conditions, the cells were washed thoroughly in phosphate-buffered saline (PBS) and fixed on ice for 30 minutes in 3.7% formaldehyde before washed in PBS and mounted with Slowfade Antifade Kit (Molecular Probes, PoortGebouw, The Netherlands) according to manufacturers instructions. Counterstaining of the cells with propidium iodide (PI) (Sigma) was carried out by adding PI to a final concentration of 5 μg/ml to the cells for 15 minutes with the test substances. Counterstaining with Hoechst 33258 (Sigma) took place following formaldehyde fixation by incubation for 20 minutes at room temperature in PBS containing Hoechst 33258 (2.5 μg/ml). No detergent was used.

The cells were examined by fluorescence microscopy on an Olympus BX61 microscope. Image analysis was performed using MetaMorph 4.6 (Universal Imaging Corporation) and Adobe Photoshop 4.0 (Adobe Systems Inc.).

The image analysis of the IMR-90 cells indicated a clearly different intracellular distribution of the cleavable PNA prodrugs PNA-4 and PNA-5 as compared to the non-cleavable PNA drugs PNA-2 and PNA-3. The cleavable prodrugs appeared to be nuclear associated although without full nuclear localisation. Figure 1 shows the images of the IMR-90 cell-uptake of the PNA-3 drug and the PNA-5 prodrug compared to the control. REFERENCES

1. Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O., Science (1991 ) 254, 1497-1500. 2. Egholm, M., Buchardt, O., Christensen, L., Behrens, C, Freier, S. M., Driver, D.A., Berg, R.H., Kim, S.K., Norden, B., Nielsen, P.E., Nature (1993) 365, 566-568.

3. Demidov, V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, O. SRnnichsen, H. S., Nielsen, P.E., Biochem. Pharmacol. (1994) 48, 1310-1313.

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

5. Hanvey J.C, Peffer N.J., Bisi J.E., Thomson S.A., Cadilla R., Josey J.A., Ricca D.J., Hassman C.F., Bonham M.A., Au K.G., Science (1992) 258 (5087),1481-5. 6. Knudsen, H. and Nielsen, P.E., Nucleic Acids Res. (1996) 24, 494-500.

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8(2):84-7. H .Brugidou, J., Legrand, Ch., Mery, J., Rabie, A., Biochemical and biophysical research communications, (1995) 214 (2), 685-693. 12. Falnes P.O., Wesche J., Olsnes S., Biochemistry (2001 ) 10 (40, 14),

4349-58. 13.Zanta M.A., Belguise-Valladier P., Behr J.P., Proc Natl Acad Sci U S A,

(1999) 96(1 ), 91-6. 14. Lindgren M., Gallet X., Soomets U., Hallbhnk M., Brakenhielm E., Pooga M., Brasseur R., Langel U., Bioconjug Chem; (2000) H(5), 619-26.

15. Magzoub M., Kilk K., Eriksson L.E., Langel U., Graslund A., Biochim

Biophys Ada; (2001 ) 1512(1 ), 77-89. 16. Pooga M., Hallbhnk M., Zorko M., Langel U., FASEB J, (1998) 12(1) 67- 77. 17. Pooga, M., Soomets, U., Hallbhnk, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J., Xu, X., Wiesenfeld-Hallin, Z., HRkfelt, T, Bartfai, T., Langel, U., Nature Biotechnology (1998) 16, 857-61. 18. Merrifield B., Science, (1986) 232 (4748), 341-347.

19. Berge, S. M., Bighley L.D., Monkhouse D.C., Pharmaceutical Science

(1977) 66, 1-19. 20.Gilman,H., Brown, G.E., Webb, F.J., Spatz, S.M., J.Amer.Chem.Soc, (1940) 62, 977. 21. Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K.H., Hansen, H.F., Koch, T., Egholm, M., Burchardt, O., Nielsen, P.E., Coull, J., Berg, R.H., J.Peptide Sci. (1995) 3, 175-183. 22. Lohse, J., Nielsen, P.E., Harrit, N., Dahl, O., Bioconjugate Chem., (1997) 8, 503-509. 23. Wong, Shan S. Chemistry of Protein Conjugation and Cross-linking, CRC Press LLC 1993.

Claims

1. A Peptide Nucleic Acid (PNA) prodrug, of formula (I)

Q - L E-L₂ - PNA (I)

wherein Q is a carrier moiety; E is a cleavable moiety;

Li and L₂ are, independently, a linker or a bond; and PNA is a peptide nucleic acid oligomer.

2. A compound of claim 1 , wherein E is an amino acid of the formula (II)

- HN - A - B - CO - (II)

in which the A - B bond is sensitive to cleavage by an enzyme.

3. A compound of claim 2, wherein the A - B bond is an ester bond.

4. A compound of claim 3, wherein - A - B - is a group of formula (III)

(III)

wherein X and Y, independently, are an alkylene, a C₂._n-alkenylene, a C₂._n-alkynylene, a phenylene, or a benzylidene group.

5. A compound of claim 2 to 4, wherein • -A - B- - is a compound of formula (IV)

(IV)

6. A compound of claim 1 - 5, wherein Q is a lipid, a fatty acid, a cell penetrating peptide, a peptidomimetic or a hormone.

7. An amino acid of the formula (V)

R¹ - HN - A - B - CO - R² (V)

R³ is an alkyl, a C₂-_n'-alkenyl, a C₂-n'-alkynyl, an aryl, or an alkylaryl, R⁴ is H, F, Cl, or Br, and R⁵ is H, or -SO₃H.

8. An amino acid of claim 7 in which the A - B bond is an ester bond.

9. An amino acid of claim 7 or 8 in which the - A - B - is a group of formula (III)

(III) in which X and Y, independently, are an alkylene group, a C₂-_n- alkenylene, a C₂-_n-alkynylene, a phenylene, or a benzylidene group.

10. An amino acid of claim 7 or 9 wherein •••A - B- - is a compound of formula (IV)

11. A method of synthesising PNA prodrugs of claims 1-6 comprising a cleavable moiety of claim 7-10.

12. A method of claim 11 comprising the following steps: a) The coupling of monomers onto a polymeric support, which is provided with an anchoring group, using a process conventionally used in solid-phase synthesis, b) cleaving off the protective group of the last amino acid or monomer of the PNA, using a suitable reagent, c) coupling onto the PNA a compound containing the cleavable monomer of claim 7-10 protected by Boc or a similar solid phase protecting group, d) cleaving off the temporary protective group by means of a suitable reagent, e) coupling on the cell penetrating carrier moiety using continuous solid phase synthesis or fragment coupling f) cleaving off the protective groups by means of a suitable reagent, g) cleaving off the compound of the invention from the polymeric support using a suitable reagent conventionally used in peptide chemistry.

13. Use of a PNA prodrug according to any of claims 1 to 10 in the manufacture of a medicament for the treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or for disinfecting non-living objects, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools, and the like.

14. Use of a PNA prodrug according to any of claims 1 to 10 in the manufacture of a composition for the treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or for disinfecting non-living objects, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools, and the like.

15. A pharmaceutical composition comprising, as an active ingredient, a compound according to any one of the preceding compound claims 1 to 10 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.

16. A composition according to claim 15 in unit dosage form, comprising from about 0.05 to about 100 mg, preferably from about 0.1 to about 50 mg of the compound according to any one of the preceding compound claims 1-10 or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition according to any one of the claims 15 to 16 for oral, nasal, transdermal, pulmonal, or parenteral administration.

18. A pharmaceutical composition according to claim 15 to 17 for the treatment or prevention of bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune and immunological disorders, or treatment of non-living objects, the composition comprising, as an active ingredient, a compound according to any one of the preceding compound claims 1 to 10 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.

19. A method of treating a disease selected from bacterial, viral, protozoal, and fungal infections, cancer, metabolic diseases, cardiovascular diseases, autoimmune or immunological disorders comprising administering to a patient in need thereof an efficient amount of a compound of claim 1 to 10, the method comprising administering to a subject in need thereof an effective amount of a compound according to any one of the preceding compound claims 1-10 or a pharmaceutically acceptable salt thereof, or of a composition according to any one of the preceding composition claims.

20. The method according to claim 19, wherein the effective amount of the compound according to any one of the preceding compound claims 1 to 10 or a pharmaceutically acceptable salt or ester thereof is in the range of from about 0.05 to about 100 mg per day, preferably from about 0.1 to about 50 mg per day.