Patent Description:
Skin aging has received considerable attention since the signs of aging are most visible in the skin. Skin aging begins in their middle or late twenties with the reduction of collagen and elastin in the skin to result in dry and low elastic skin and even wrinkles. Obesity is a kind of inflammation reaction caused by the decline in blood circulation came from excessively deposited internal fat. Internal fat on blood vessel inhibits blood circulation and secretion of various hormones to promote aging in the whole body including the skin. In that sense, health conditions and diseases linked to obesity have to be monitored to get healthy and beautiful skin.

The biosynthesis and degradation of fatty acids are well regulated according to the physiological conditions to meet the demand of the body. Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA, which is the rate-determining step in the first stage of fatty acid biosynthesis.

ACC has a function of controlling metabolism of fatty acids in two ways. The most important function of ACC is to provide the malonyl-CoA substrate as a new building block in its active state for the fatty acid biosynthesis. Another function is to block the oxidation of fatty acids in mitochondria through inhibition of acyl group transfer of fatty acids.

In human, two main isoforms of ACC are expressed, acetyl-CoA carboxylase <NUM> (ACC1, ACACA, acetyl-CoA carboxylase alpha) and acetyl-CoA carboxylase <NUM> (ACC2, ACACB, acetyl-CoA carboxylase beta). Two ACCs have different functions each other, i.e., ACC1 maintains regulation of fatty acid synthesis whereas ACC2 mainly regulates fatty acid oxidation.

ACCs regulating biosynthesis and oxidation of fatty acids are potential targets for the treatment of many diseases such as new antibiotics utilizing the structure differences of bacteria and human ACCs, metabolic syndrome of diabetics and obesity, lipogenesis related growth inhibitors of cancer cell, and so on [<NPL>); <NPL>)].

Among them, a study on the ACC2-/- mutant mice has attracted lots of attention, where ACC2-deficient mice had lower level of fat with a higher fatty acid oxidation rate, lost or maintained body weight in spite of more food consumption, and had reduced risk of diabetes [<NPL>)]. These results suggested the possibility of ACC2 inhibitors to have a therapeutic effect on obesity and diabetes. In addition, treatment of the inhibitors to the skin may expect the effect of fat removal and eventually the prevention of obesity in the skin and the improvement of skin aging.

Considering the significance of obesity in skin aging process, it is very interesting and necessary to develop ACC2 inhibitors or the pharmaceuticals or cosmetics based on the mechanism of ACC2 expression, which may improve and prevent skin aging condition.

Pre-mRNA: Genetic information is carried on DNA (<NUM>-deoxyribose nucleic acid). DNA is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. Mammalian pre-mRNA usually consists of exons and introns, and exon and intron are interconnected to each other as schematically provided below. Exons and introns are numbered as exemplified in the drawing below.

Splicing of Pre-mRNA: Pre-mRNA is processed into mRNA following deletion of introns by a series of complex reactions collectively called "splicing" which is schematically summarized in the diagram below [<NPL>); <NPL>); <NPL>)].

Splicing is initiated by forming "spliceosome E complex" (i.e. early spliceosome complex) between pre-mRNA and splicing adapter factors. In "spliceosome E complex", U1 binds to the junction of exon N and intron N, and U2AF<NUM> binds to the junction of intron N and exon (N+<NUM>). Thus the junctions of exon/intron or intron/exon are critical to the formation of the early spliceosome complex. "Spliceosome E complex" evolves into "spliceosome A complex" upon additional complexation with U2. The "spliceosome A complex" undergoes a series of complex reactions to delete or splice out the intron to adjoin the neighboring exons.

Ribosomal Protein Synthesis: Proteins are encoded by DNA (<NUM>-deoxyribose nucleic acid). In response to cellular stimulation or spontaneously, DNA is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. The introns of pre-mRNA are enzymatically spliced out to yield mRNA (messenger ribonucleic acid), which is then translocated into the cytoplasm. In the cytoplasm, a complex of translational machinery called ribosome binds to mRNA and carries out the protein synthesis as it scans the genetic information encoded along the mRNA [<NPL>); <NPL>)].

Antisense Oligonucleotide (ASO): An oligonucleotide binding to nucleic acid including DNA, mRNA and pre-mRNA in a sequence specific manner (i.e. complementarily) is called antisense oligonucleotide (ASO).

If an ASO tightly binds to an mRNA in the cytoplasm, for example, the ASO may be able to inhibit the ribosomal protein synthesis along the mRNA. ASO needs to be present within the cytoplasm in order to inhibit the ribosomal protein synthesis of its target protein.

Antisense Inhibition of Splicing: If an ASO tightly binds to a pre-mRNA in the nucleus, the ASO may be able to inhibit or modulate the splicing of pre-mRNA into mRNA. ASO needs to be present within the nucleus in order to inhibit or modulate the splicing of pre-mRNA into mRNA. Such antisense inhibition of splicing produces an mRNA or mRNAs lacking the exon targeted by the ASO. Such mRNA(s) is called "splice variant(s)", and encodes protein(s) smaller than the protein encoded by the full-length mRNA.

In principle, splicing can be interrupted by inhibiting the formation of "spliceosome E complex". If an ASO tightly binds to a junction of (<NUM>' → <NUM>') exon-intron, i.e. "<NUM>' splice site", the ASO blocks the complex formation between pre-mRNA and factor U1, and therefore the formation of "spliceosome E complex". Likewise, "spliceosome E complex" cannot be formed if an ASO tightly binds to a junction of (<NUM>' → <NUM>') intron-exon, i.e. "<NUM>' splice site".

<NUM>' splice site and <NUM>' splice site are schematically illustrated in the drawing provided below.

Unnatural Oligonucleotides: DNA or RNA oligonucleotides are susceptible to degradation by endogenous nucleases, limiting their therapeutic utility. To date, many types of unnatural (naturally non-occurring) oligonucleotides have been developed and studied intensively [<NPL>)]. Some of them show extended metabolic stability compared to DNA and RNA. Provided below are the chemical structures for a few of representative unnatural oligonucleotides. Such oligonucleotides predictably bind to a complementary nucleic acid as DNA or RNA does.

Phosphorothioate Oligonucleotide: Phosphorothioate oligonucleotide (PTO) is a DNA analog with one of the backbone phosphate oxygen atoms replaced with a sulfur atom per monomer. Such a small structural change made PTO comparatively resistant to degradation by nucleases [<NPL>)].

Reflecting the structural similarity in the backbone of PTO and DNA, they both poorly penetrate the cell membrane in most mammalian cell types. For some types of cells abundantly expressing transporter(s) of DNA, however, DNA and PTO show good cell penetration. Systemically administered PTOs are known to readily distribute to the liver and kidney [<NPL>)].

In order to facilitate PTO's cell penetration in vitro, lipofection has been popularly practiced. However, lipofection physically alters the cell membrane, causes cytotoxicity, and therefore would not be ideal for long term in vivo therapeutic use.

Over the past <NUM> years, antisense PTOs and variants of PTOs have been clinically evaluated to treat cancers, immunological disorders, metabolic diseases, and so on [<NPL>);<NPL>)]. Many of such antisense drug candidates have not been successfully developed partly due to PTO's poor cell penetration. In order to overcome the poor cell penetration, PTO needs to be administered at high dose for therapeutic activity. However, PTOs are known to be associated with dose-limiting toxicity including increased coagulation time, complement activation, tubular nephropathy, Kupffer cell activation, and immune stimulation including splenomegaly, lymphoid hyperplasia, mononuclear cell infiltration [<NPL>)].

Many antisense PTOs have been found to show due clinical activity for diseases with a significant contribution from the liver or kidney. Mipomersen is a PTO analog which inhibits the synthesis of apoB-<NUM>, a protein involved in LDL cholesterol transport. Mipomersen manifested due clinical activity in atherosclerosis patients most likely due to its preferential distribution to the liver [<NPL>)]. ISIS-<NUM> is a PTO antisense analog inhibiting the synthesis of protein tyrosine phosphatase 1B (PTP1B), and was found to show therapeutic activity in type II diabetes patients.

Locked Nucleic Acid: In locked nucleic acid (LNA), the backbone ribose ring of RNA is structurally constrained to increase the binding affinity for RNA or DNA. Thus, LNA may be regarded as a high affinity DNA or RNA analog [<NPL>)].

Phosphorodiamidate Morpholino Oligonucleotide: In phosphorodiamidate morpholino oligonucleotide (PMO), the backbone phosphate and <NUM>-deoxyribose of DNA are replaced with phosphoramidate and morpholine, respectively [<NPL>)]. Whilst the DNA backbone is negatively charged, the PMO backbone is not charged. Thus the binding between PMO and mRNA is free of electrostatic repulsion between the backbones, and tends to be stronger than that between DNA and mRNA. Since PMO is structurally very different from DNA, PMO wouldn't be recognized by the hepatic transporter recognizing DNA. PMO may exhibit a different tissue distribution than PTO, but PMO, like PTO, doesn't readily penetrate the cell membrane.

Peptide Nucleic Acid: Peptide nucleic acid (PNA) is a polypeptide with N-(<NUM>-aminoethyl)glycine as the unit backbone, and was discovered by Dr. Nielsen and colleagues [<NPL>)]. The chemical structure and abbreviated nomenclature of PNA are illustrated in the drawing provided below. Like DNA and RNA, PNA also selectively binds to complementary nucleic acid. In binding to complementary nucleic acid, the N-terminus of PNA is regarded as equivalent to the "<NUM>'-end" of DNA or RNA, and the C-terminus of PNA as equivalent to the "<NUM>'-end" of DNA or RNA.

Like PMO, the PNA backbone is not charged. Thus the binding between PNA and RNA tends to be stronger than the binding between DNA and RNA. Since PNA is markedly different from DNA in the chemical structure, PNA wouldn't be recognized by the hepatic transporter(s) recognizing DNA, and would show a tissue distribution profile different from that of DNA or PTO. However, PNA also poorly penetrates the mammalian cell membrane [<NPL>)].

Modified Nucleobases to Improve Membrane Permeability of PNA: PNA was made highly permeable to mammalian cell membrane by introducing modified nucleobases with a cationic lipid or its equivalent covalently attached thereto. The chemical structures of such modified nucleobases are provided below. Such modified nucleobases of cytosine, adenine, and guanine were found to predictably and complementarily hybridize with guanine, thymine, and cytosine, respectively [PCT Appl. No. <CIT>; <CIT>; <CIT>].

Incorporation of such modified nucleobases onto PNA resembles situations of lipofection. By lipofection, oligonucleotide molecules with phosphate backbone are wrapped with cationic lipid molecules such as lipofectamine, and such lipofectamine/oligonucleotide complexes tend to penetrate membrane rather easily as compared to naked oligonucleotide molecules.

In addition to good membrane permeability, those PNA derivatives were found to possess ultra-strong affinity for complementary nucleic acid. For example, introduction of <NUM> to <NUM> modified nucleobases onto <NUM>- to <NUM>-mer PNA derivatives easily yielded a Tm gain of <NUM> or higher in duplex formation with complementary DNA. Such PNA derivatives are highly sensitive to a single base mismatch. A single base mismatch resulted in a Tm loss of <NUM> to <NUM> depending on the type of modified base as well as PNA sequence.

Small Interfering RNA (siRNA): Small interfering RNA (siRNA) refers to a double stranded RNA of <NUM>-<NUM> base pairs [<NPL>)]. The antisense strand of siRNA somehow interacts with proteins to form an "RNA-induced Silencing Complex" (RISC). Then the RISC binds to a certain portion of mRNA complementary to the antisense strand of siRNA. The mRNA complexed with the RISC undergoes cleavage. Thus siRNA catalytically induces the cleavage of its target mRNA, and consequently inhibits the protein expression by the mRNA. The RISC does not always bind to the full complementary sequence within its target mRNA, which raises concerns relating to off-target effects of an siRNA therapy. Like other classes of oligonucleotide with DNA or RNA backbone, siRNA possesses poor cell permeability and therefore tends to show poor in vitro or in vivo therapeutic activity unless properly formulated or chemically modified to have good membrane permeability.

ACC siRNA: The mixture of ACC1 siRNA and ACC2 siRNA was reported to inhibit the expression of ACC1 and ACC2 mRNAs and proteins in glioblastoma cancer cell line following a lipofection at <NUM> each [<NPL>)]. These results may be useful to the study of ACC related lipogenic cancer metastasis.

<CIT>, in the name of Isis Pharmaceutical Inc. describes antisense oligonucleotides that are complementary to part of the ACC2 pre-mRNA and which may be synthesized as peptide nuclide acids. These target certain part of the human ACC2 pre-mRNA. The document refers to the possibility of perturbing splice site selection, but the majority of the specific antisense oligonucleotides taught in this document are gapmers. <CIT>, in the name of Olipas Corp, describes the design of peptide nucleic acids comprising the nucleobase analogues of Formulae II, III and IV. Neither document suggests the design of antisense oligonucleotides targeting the exon <NUM>/intron <NUM> junction of the human ACC2 pre-mRNA.

Since obesity has a profound effect on skin aging, health conditions and diseases linked to obesity have to be monitored to get healthy and beautiful skin.

A study on the ACC2-/- mutant mice with respect to obesity has attracted lots of attention. In addition, although ACCs siRNA were reported to inhibit the expression of ACCs mRNAs and proteins in cancer cell line, siRNAs are too expensive to manufacture and develop as anti-aging agent for skin to say nothing of their delivery challenge into the skin. Therefore, it is necessary to develop the pharmaceuticals or cosmetics based on the mechanism of ACC2 expression, which may improve and prevent skin aging condition.

The present invention provides a peptide nucleic acid (PNA) derivative represented by Formula I, or a pharmaceutically acceptable salt thereof, for inducing exon skipping within human ACC2 pre-mRNA:
<CHM>
wherein,.

The compound of Formula I induces the skipping of "exon <NUM>" in the human ACC2 pre-mRNA, yields the human ACC2 mRNA splice variant(s) lacking "exon <NUM>", and therefore is useful to inhibit the functional activity of the gene transcribing the human ACC2 pre-mRNA.

The condition that "n is an integer between <NUM> and <NUM>" literally means that n is an integer selectable from a group of integers of <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

The chemical structures of natural or unnatural nucleobases in the PNA derivative of Formula I are exemplified in <FIG>. Natural (i.e. naturally occurring) or unnatural (naturally non-occurring) nucleobases of this invention comprise but are not limited to the nucleobases provided in <FIG>. Provision of such unnatural nucleobases is to illustrate the diversity of allowable nucleobases, and therefore should not be interpreted to limit the scope of the present invention.

The substituents adopted to describe the PNA derivative of Formula I are exemplified in <FIG>. <FIG> provides examples for substituted or non-substituted alkyl radicals. Substituted or non-substituted alkylacyl and substituted or non-substituted aryl acyl radicals are exemplified in <FIG>. <FIG> illustrates examples for substituted or non-substituted alkylamino, substituted or non-substituted arylamino, substituted or non-substituted aryl, substituted or non-substituted alkylsulfonyl or arylsulfonyl, and substituted or non-substituted alkylphosphonyl or arylphosphonyl radicals. <FIG> provides examples for substituted or non-substituted alkyloxycarbonyl or aryloxycarbonyl, substituted or non-substituted alkyl aminocarbonyl or arylaminocarbonyl radicals. In <FIG> are provided examples for substituted or non-substituted alkylaminothiocarbonyl, substituted or non-substituted arylaminothiocarbonyl, substituted or non-substituted alkyloxythiocarbonyl, and substituted or non-substituted aryloxythiocarbonyl radicals. Provision of such exemplary substituents is to illustrate the diversity of allowable substituents, and therefore should not be interpreted to limit the scope of the present invention. A skilled person in the field may easily figure out that oligonucleotide sequence is the overriding factor for sequence specific binding of oligonucleotide to the target pre-mRNA sequence over substituents in the N-terminus or C-terminus.

The compound of Formula I tightly binds to the complementary DNA as exemplified in the prior art [<CIT>]. The duplex between the PNA derivative of Formula I and its full-length complementary DNA or RNA possesses a Tm value too high to be reliably determined in aqueous buffer. The PNA compound of Formula I yields high Tm values with complementary DNAs of shorter length.

The compound of Formula I complementarily binds to the <NUM>' splice site of "exon <NUM>" of the human ACC2 pre-mRNA. [NCBI Reference Sequence: NG_046907]. The <NUM>-mer sequence of [(<NUM>'→<NUM>') GCCAUUUCGUCAGUAU] spans the junction of "exon <NUM>" and "intron <NUM>" in the human ACC2 pre-mRNA, and consists of <NUM>-mer from " exon <NUM>" and <NUM>-mer from " intron <NUM>". Thus the <NUM>-mer pre-mRNA sequence may be conventionally denoted as [(<NUM>'→<NUM>') GCCAUUUC | gucaguau], wherein the exon and intron sequence are provided as "capital " and "small" letters, respectively, and the exon-intron junction is expressed with " | ". The conventional denotation for pre-mRNA is further illustrated by a <NUM>-mer sequence of [(<NUM>'→<NUM>') GGAAGAGGCCAUUUC | gucaguaucuccuuc] spanning the junction of "exon <NUM>" and "intron <NUM>" in the human ACC2 pre-mRNA.

The compound of Formula I tightly binds to the target <NUM>' splice site of the human ACC2 pre-mRNA transcribed from the human ACC2 gene, and interferes with the formation of "spliceosome early complex" to yield ACC2 mRNA splice variant(s) lacking "exon <NUM>" (exon <NUM> skipping).

The strong RNA affinity allows the compound of Formula I to induce the skipping of ACC2 "exon <NUM>", even when the PNA derivative possesses one or two mismatches with the target <NUM>' splice site in the ACC2 pre-mRNA. Similarly the PNA derivative of Formula I may still induce the skipping of ACC2 "exon <NUM>" in a ACC2 mutant pre-mRNA possessing one or two SNPs (single nucleotide polymorphism) in the target splice site.

The compound of Formula I possesses good cell permeability and can be readily delivered into cell as "naked" oligonucleotide as exemplified in the prior art [<CIT>]. Thus the compound of this invention induces the skipping of "exon <NUM>" in the ACC2 pre-mRNA, and yields ACC2 mRNA splice variant(s) lacking ACC2 "exon <NUM>" in cells treated with the compound of Formula I as "naked" oligonucleotide. The compound of Formula I does not require any means or formulations for delivery into cell to potently induce the skipping of the target exon in cells. The compound of Formula I readily induces the skipping of ACC2 "exon <NUM>" in cells treated with the compound of this invention as "naked" oligonucleotide at sub-femtomolar concentration.

Owing to the good cell or membrane permeability, the PNA derivative of Formula I can be topically administered as "naked" oligonucleotide to induce the skipping of ACC2 "exon <NUM>" in the skin. The compound of Formula I does not require a formulation to increase trans-dermal delivery into target tissue for the intended therapeutic or biological activity. Usually the compound of Formula I is dissolved in water and co-solvent, and topically or trans-dermally administered at subpicomolar concentration to elicit the desired therapeutic or biological activity in target skin. The compound of this invention does not need to be heavily or invasively formulated to elicit the topical therapeutic activity. Nevertheless, the PNA derivative of Formula I can be formulated with cosmetic ingredients or adjuvants as topical cream or lotion. Such topical cosmetic cream or lotion may be useful to treat skin aging.

The compound of the present invention can be topically administered to a subject at a therapeutically or biologically effective concentration ranging from <NUM> aM to higher than <NUM>, which would vary depending on the dosing schedule, conditions or situations of the subject, and so on.

The PNA derivative of Formula I can be variously formulated including but not limited to injections, nasal spray, transdermal patch, and so on. In addition, the PNA derivative of Formula I can be administered to the subject at therapeutically effective dose and the dose of administration can be diversified depending on indication, administration route, dosing schedule, conditions or situations of the subject, and so on.

The compound of Formula I may be used as combined with a pharmaceutically acceptable acid or base including but not limited to sodium hydroxide, potassium hydroxide, hydrochloric acid, methanesulfonic acid, citric acid, trifluoroacetic acid, and so on.

The PNA derivative of Formula I or a pharmaceutically acceptable salt thereof can be administered to a subject in combination with a pharmaceutically acceptable adjuvant including but not limited to citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethyleneglycol, polypropyleneglycol, ethanol, isopropanol, sodium bicarbonate, distilled water, preservative(s), and so on.

Of higher interest is a PNA derivative of Formula I, or a pharmaceutically acceptable salt thereof:
wherein,.

Of specific interest is a PNA derivative of Formula I which is selected from the group of compounds provided below (Hereinafter referred to as ASOs <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, respectively), or a pharmaceutically acceptable salt thereof:.

<FIG> collectively and unambiguously provides the chemical structures for the PNA monomers abbreviated as A, G, T, C, C(pOq), A(p), and G(p). As discussed in the prior art [<CIT>], C(pOq) is regarded as a "modified cytosine" PNA monomer due to its hybridization for "guanine". A(p) is taken as "modified adenine" PNA monomers due to their hybridization for "thymine", and G(p) is taken as "modified guanine" PNA monomers due to their hybridization for "cytosine". In addition, in order to illustrate the abbreviations employed for such PNA derivatives, the chemical structure of ASO <NUM> "(N → C) CTG(<NUM>)-ACG(<NUM>)-AA(<NUM>)A-TG(<NUM>)G-C(1O2)C-NH<NUM>" is provided in <FIG>.

ASO <NUM> is equivalent to the DNA sequence of "(<NUM>' → <NUM>') CTG-ACG-AAA-TGG-CC" for complementary binding to pre-mRNA. The <NUM>-mer PNA has a <NUM>-mer complementary overlap with the <NUM>-mer sequence marked "bold" and "underlined" within the <NUM>-mer RNA sequence of [(<NUM>' → <NUM>') GGAAGAGGCCAUUUC | gucaguaucuccuuc] spanning the junction of "exon <NUM>" and "intron <NUM>" in the human ACC2 pre-mRNA.

In some embodiments, the present invention provides the peptide nucleic acid derivative set out above, or a pharmaceutically acceptable salt thereof for use in a method of treating conditions or disorders associated with human ACC2 gene transcription in a subject, comprising administering to the subject the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides the peptide nucleic acid derivative defined above, or a pharmaceutically acceptable salt thereof for use in a method of treating skin aging in a subject, comprising administering to the subject the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceutical composition for treating conditions or disorders associated with human ACC2 gene transcription, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a cosmetic composition for treating conditions or disorders associated with human ACC2 gene transcription, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceutical composition for treating skin aging, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a cosmetic composition for treating skin aging, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

Conditions or disorders associated with human ACC2 gene transcription can be treated by administering a PNA derivative of Formula I or a pharmaceutically acceptable salt thereof.

Skin aging can be treated by administering a PNA derivative of Formula I or a pharmaceutically acceptable salt thereof.

PNA oligomers were synthesized by solid phase peptide synthesis (SPPS) based on Fmoc-chemistry according to the method disclosed in the prior art [<CIT>; <CIT>] with minor but due modifications. Fmoc is {(<NUM>-fluorenyl)methyloxy}carbonyl. The solid support employed in this study was H-Rink Amide-ChemMatrix purchased from PCAS BioMatrix Inc. (Quebec, Canada). Fmoc-PNA monomers with a modified nucleobase were synthesized as described in the prior art [<CIT>] or with minor modifications. Such Fmoc-PNA monomers with a modified nucleobase and Fmoc-PNA monomers with a naturally occurring nucleobase were used to synthesize the PNA derivatives of the present invention. PNA oligomers were purified by C<NUM>-reverse phase HPLC (water/acetonitrile or water/methanol with <NUM>% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

Scheme <NUM> illustrates a typical monomer elongation cycle adopted in the SPPS of this study, and the synthetic details are provided as below. To a skilled person in the field, however, there are lots of minor variations obviously possible in effectively running such SPPS reactions on an automatic peptide synthesizer or manual peptide synthesizer. Each reaction step in Scheme <NUM> is briefly provided as follows.

[Activation of H-Rink-ChemMatrix Resin] When the amine on the resin was not protected with Fmoc, <NUM> mmol (ca <NUM> resin) of the ChemMatrix resin in <NUM> <NUM>% piperidine/DMF was vortexed in a libra tube for <NUM>, and the DeFmoc solution was filtered off. The resin was washed for <NUM> sec each in series with <NUM> methylene chloride (MC), <NUM> dimethylformamide (DMF), <NUM> MC, <NUM> DMF, and <NUM> MC. The resulting free amines on the solid support were subjected to coupling with an Fmoc-PNA monomer.

[DeFmoc] When the amine on the resin was protected with Fmoc, the suspension of <NUM> mmol (ca <NUM>) of the resin in <NUM> <NUM>% piperidine/DMF was vortexed for <NUM>, and the DeFmoc solution was filtered off. The resin was washed for <NUM> sec each in series with <NUM> MC, <NUM> DMF, <NUM> MC, <NUM> DMF, and <NUM> MC. The resulting free amines on the solid support were immediately subjected to coupling with an Fmoc-PNA monomer.

[Coupling with Fmoc-PNA Monomer] The free amines on the solid support were coupled with an Fmoc-PNA monomer as follows. <NUM> mmol of PNA monomer, <NUM> mmol HBTU, and <NUM> mmol DIEA were incubated for <NUM> in <NUM> anhydrous DMF, and added to the resin with free amines. The resin solution was vortexed for <NUM> hour and the reaction medium was filtered off. Then the resin was washed for <NUM> sec each in series with <NUM> MC, <NUM> DMF, and <NUM> MC. The chemical structures of Fmoc-PNA monomers with a modified nucleobase used in this invention are provided in <FIG>. The Fmoc-PNA monomers with a modified nucleobase are provided in <FIG> should be taken as examples, and therefore should not be taken to limit the scope of the present invention. A skilled person in the field may easily figure out a number of variations in Fmoc-PNA monomers to synthesize the PNA derivative of Formula I.

[Capping] Following the coupling reaction, the unreacted free amines were capped by shaking for <NUM> in <NUM> capping solution (<NUM>% acetic anhydride and <NUM>% <NUM>,<NUM>-leutidine in DMF). Then the capping solution was filtered off and washed for <NUM> sec each in series with <NUM> MC, <NUM> DMF, and <NUM> MC.

[Introduction of "Fethoc-" Radical in N-Terminus] "Fethoc-" radical was introduced to the N-terminus by reacting the free amine on the resin with "Fethoc-OSu" by the following method. The suspension of the resin in the solution of <NUM> mmol of Fethoc-OSu and <NUM> mmol DIEA in <NUM> anhydrous MDF was vortexed for <NUM> hr, and the solution was filtered off. The resin was washed for <NUM> sec each in series with <NUM> MC, <NUM> DMF, and <NUM> MC. The chemical structure of "Fethoc-OSu" [CAS No. <NUM>-<NUM>-<NUM>, C<NUM>H<NUM>NO<NUM>, MW <NUM>] used in the present invention is provided as follows.

[Cleavage from Resin] PNA oligomers bound to the resin were cleaved from the resin by shaking for <NUM> hours in <NUM> cleavage solution (<NUM>% tri-isopropylsilane and <NUM>% water in trifluoroacetic acid). The resin was filtered off and the filtrate was concentrated under reduced pressure. The resulting residue was triturated with diethyl ether and the resulting precipitate was collected by filtration for purification by reverse phase HPLC.

[HPLC Analysis and Purification] Following a cleavage from resin, the crude product of a PNA derivative was purified by C<NUM>-reverse phase HPLC eluting water/acetonitrile or water/methanol (gradient method) containing <NUM>% TFA. <FIG> and <FIG> are exemplary HPLC chromatograms for "ASO <NUM>" before and after HPLC purification, respectively.

In order to complementarily target the <NUM>' splice site of "exon <NUM>" in the human ACC2 pre-mRNA, PNA derivatives of this invention were prepared according to the synthetic procedures provided above or with minor modifications. Provision of such PNA derivatives targeting the human ACC2 pre-mRNA is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

Table <NUM> provides PNA derivatives complementarily targeting the <NUM>' splice site of "exon <NUM>" in the human ACC2 pre-mRNA read out from the human ACC2 gene [NCBI Reference Sequence: NG_046907] along with structural characterization data by mass spectrometry. Provision of the peptide nucleic acid derivatives of the present invention in Table <NUM> is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

"ASO <NUM>" has a <NUM>-mer complementary overlap with the <NUM>-mer sequence marked "bold" and "underlined" within the <NUM>-mer RNA sequence of [(<NUM>' → <NUM>') GGAAGAGGCCAUUUC | gucaguaucuccuuc] spanning the junction of "exon <NUM>" and "intron <NUM>" in the human ACC2 pre-mRNA. Thus "ASO <NUM>" possesses a <NUM>-mer overlap with "exon <NUM>" and a <NUM>-mer overlap with "intron <NUM>" within the human ACC2 pre-mRNA.

The PNA derivatives of Formula I were evaluated for their binding affinity for <NUM>-mer DNAs complementarily targeting either the N-terminal or C-terminal. The binding affinity was assessed by Tm value for the duplex between PNA and <NUM>-mer complementary DNA. The duplex between PNA derivatives and fully complementary DNAs show Tm values too high to be reliably determined in aqueous buffer solution, since the buffer solution tends to boil during the Tm measurement. Tm values for full length PNAs can be predicted and compared based on the Tm value for the duplex between PNA and <NUM>-mer complementary DNA.

Tm values were determined on a UV/Vis spectrometer as follows. A mixed solution of <NUM> PNA oligomer and <NUM> complementary <NUM>-mer DNA in <NUM> aqueous buffer (pH <NUM>, <NUM> sodium phosphate, <NUM> NaCl) in <NUM> polypropylene falcon tube was incubated at <NUM> for a few minute and slowly cooled down to ambient temperature. Then the solution was transferred into a <NUM> quartz UV cuvette equipped with an air-tight cap, and the cuvette was mounted on an Agilent <NUM> UV/Visible spectrophotometer. The absorbance changes at <NUM> were recorded with increasing the temperature of the cuvette by either <NUM> or <NUM> per minute. From the absorbance vs temperature curve, the temperature showing the largest increase rate in absorbance was read out as the Tm between PNA and <NUM>-mer DNA. The DNAs for Tm measurement were purchased from Bioneer (www. com, Dajeon, Republic of Korea) and used without further purification.

Observed Tm values of the PNA derivatives of Formula I are very high for a complementary binding to <NUM>-mer DNA as provided in Table <NUM>.

For example, "ASO <NUM>" showed a Tm value of <NUM> for the duplex with the <NUM>-mer complementary DNA targeting the N-terminal <NUM>-mer in the PNA as marked "bold" and "underlined" in [(N → C) Fethoc-CTG(<NUM>)-ACG(<NUM>)-AA(<NUM>)A- TG(<NUM>)G-C(1O2)C-NH<NUM>]. In the meantime, "ASO <NUM>" showed a Tm of <NUM> for the duplex with the <NUM>-mer complementary DNA targeting the C-terminal <NUM>-mer in the PNA as marked "bold" and "underlined" in [(N → C) Fethoc-CTG(<NUM>)-ACG(<NUM>)- AA(<NUM>)A- TG(<NUM>)G-C(1O2)C-NH<NUM>].

PNA derivatives in this invention were evaluated for in vitro ACC2 antisense activities in C2C12 skeletal muscle cells by use of real-time quantitative polymerase chain reaction (RT-qPCR) and so on. The biological examples were provided as examples to illustrate the biological profiles of the PNA derivatives of Formula I, and therefore should not be interpreted to limit the scope of the current invention.

"ASO <NUM>" was evaluated for its ability to induce the skipping of ACC2 "exon <NUM>" in C2C12 cells as described below.

[Cell Culture & ASO Treatment] C2C12 cells (<NUM>×<NUM><NUM>) (Cat. No. CRL-<NUM>, ATCC) were grown in <NUM> culture dish containing DMEM medium (Dulbecco Modified Eagle Medium: DMEM) (Cat. No. <NUM>-604F, Lonza) supplemented with <NUM>% FBS (Fetal Bovine Serum) (Cat. No. <NUM>-<NUM>, GIBCO) and <NUM>% streptomycin/penicillin (Cat. No. <NUM>-<NUM>, GIBCO) under <NUM>% CO<NUM> atmosphere at <NUM>. The cells were treated either with nothing (negative control) or with an aliquot of aqueous stock solution of "ASO <NUM>" for <NUM> hours at <NUM> zM to <NUM> fM.

[RNA Extraction & Nested PCR] Total RNA was extracted using RNeasy Mini kit (Qiagen, Cat. No. <NUM>) according to the manufacturer's instructions from "ASO <NUM>" treated cells and cDNA was prepared from <NUM> ng of RNA by use of SuperScript™ III One-Step RT-PCR System (Cat. No. <NUM>-<NUM>, Invitrogen). To a mixture of <NUM> ng of RNA, <NUM> microliter of 2X Reaction Mix buffer, <NUM> microliter of SuperScript III™ RT/Platinum Taq Mix, <NUM> microliter of <NUM> (micromole conc. ) Exon <NUM> Forward Primer (<NUM>'-TTTTCCGACAAGTGCAGAG-<NUM>'), and <NUM> microliter of <NUM> Exon <NUM> Reverse Primer (<NUM>'-AACGTCCACAATGTTCAG-<NUM>') in PCR tube was added autoclaved distilled water to a total volume of <NUM> microliter. After reaction at <NUM> for <NUM> minutes and at <NUM> for <NUM> minutes, <NUM> cycles PCR process at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and at <NUM> for <NUM> minute afforded the first crude product. The mixture of <NUM> microliter of the crude product, <NUM> microliter of <NUM> Exon <NUM> Forward Primer (<NUM>'-GAG TAC TTA TAC AGC CAG G-<NUM>'), and <NUM> microliter of <NUM> Exon <NUM> Reverse Primer (<NUM>'-TTC TGA ACA TCG CGT CTG-<NUM>') was reacted, using PyroHostStart Taq Polymerase Kit (Cat. No. K-<NUM>-FCG) according to the manufacturer's instructions, at <NUM> for <NUM> minutes, and then was under PCR process at <NUM> for <NUM> seconds, at <NUM> for <NUM> minute, and at <NUM> for <NUM> seconds.

[Identification of Exon Skipping Products Electrophoresis] The PCR products (<NUM> microliter) were subjected to electrophoretic separation on a <NUM>% agarose gel. The target bands from "ASO <NUM>" treatment were collected and analyzed by Sanger Sequencing to evaluate exon skipping sequence.

[Exon Skipping Induced by "ASO <NUM>"] As can be seen in <FIG>, the cells treated with "ASO <NUM>" at <NUM> aM to <NUM> fM concentration-dependently yielded the splice variant ACC2 mRNA lacking exon <NUM>.

"ASO <NUM>" was evaluated by Real-Time qPCR for its ability to down-regulate the ACC2 mRNA formation in C2C12 as described below.

[Cell Culture & ASO Treatment] C2C12 cells (Cat. No. CRL-<NUM>, ATCC) were maintained in Dulbecco Modified Eagle Medium (DMEM, Cat. No. <NUM>-604F, Lonza) supplemented with <NUM>% Fetal Bovine Serum (Cat. No. <NUM>-<NUM>, GIBCO) and <NUM>% streptomycin/penicillin (Cat. No. <NUM>-<NUM>, GIBCO), which was grown at <NUM> and under <NUM>% CO<NUM> condition. C2C12 cells (<NUM>×<NUM><NUM>) stabilized for <NUM> hours in <NUM> culture dish were incubated for <NUM> hours with "ASO <NUM>" at <NUM> (negative control) and <NUM> zM to <NUM> fM.

[RNA Extraction & cDNA Synthesis] Total RNA was extracted using RNeasy Mini kit (Qiagen, Cat. No. <NUM>) according to the manufacturer's instructions from "ASO <NUM>" treated cells and cDNA was prepared from <NUM> ng of RNA by use of PrimeScript™ <NUM>st strand cDNA Synthesis Kit (Takara, Cat. No. 6110A). To a mixture of <NUM> ng of RNA, <NUM> microliter of random hexamer, and <NUM> microliter of dNTP (<NUM>) in PCR tube was added DEPC-treated water to a total volume of <NUM> microliter, which was reacted at <NUM> for <NUM> minutes. cDNA was synthesized by adding <NUM> microliter of PrimeScript RTase to the reaction mixture and reacting at <NUM> for <NUM> minutes and at <NUM> for <NUM> minutes, successively.

[Quantitative Real-Time PCR] In order to evaluate the expression level of human ACC2 mRNA real-time qPCR was performed with synthesized cDNA by use of Taqman probe. The mixture of cDNA, Taqman probe (Thermo, Mm01204651), IQ supermix (BioRad, Cat. No. <NUM>-<NUM>), and nuclease free water in PCR tube was under reaction by use of CFX96 Touch Real-Time system (BioRad) according to the cycle conditions specified as follows: at <NUM> for <NUM> (primary denaturation) followed by <NUM> cycles of <NUM> sec at <NUM> (denaturation) and <NUM> sec at <NUM> (annealing and polymerization). Fluorescence intensity was measured at the end of every cycle and the result of PCR was evaluated by the melting curve. After the threshold cycle (Ct) of each gene was standardized by that of GAPDH, the change of Ct was compared and analyzed.

[ACC2 mRNA Decrease by "ASO <NUM>"] As can be seen in <FIG>, compared to control experiment the amount of ACC2 mRNA was reduced at <NUM> zM to <NUM> fM treatment of "ASO <NUM>", concentration-dependently, and statistically significant <NUM>% of reduction was observed at 1fM treatment of "ASO <NUM>". (Student T-test was done to check the statistical significance of the findings).

[ACC2 mRNA Decrease by "ASO <NUM>"] As can be seen in <FIG>, the amount of ACC2 mRNA was reduced at <NUM> zM to <NUM> fM treatment of "ASO <NUM>", concentration-dependently. Compared to the control experiment, statistically significant <NUM>% and <NUM>% reduction was observed at <NUM> aM and 1fM treatment of "ASO <NUM>", respectively. (Student T-test was done to check the statistical significance of the findings).

"ASO <NUM>" was evaluated by the same method as described below.

A compound of Formula I, for example "ASO <NUM>" was formulated as a body lotion for topical application to subjects. The body lotion was prepared as described below. Given that there are lots of variations of body lotion possible, this preparation should be taken as an example and should not be interpreted to limit the scope of the current invention.

In a separate beaker, mixed substances of part A and part B were dissolved at <NUM>, respectively. Part A and part B was mixed and emulsified by use of <NUM>,<NUM> rpm homogenizer at <NUM> for <NUM> minutes. Emulsified part C was filtered through <NUM> mesh and the filtrate was added to the mixture of part A and B. The resulting mixture was emulsified by use of <NUM>,<NUM> rpm homogenizer at <NUM> for <NUM> minutes. After addition of part D to the mixture of part A, B, and C at <NUM>, the resulting mixture was emulsified by use of <NUM>,<NUM> rpm homogenizer at <NUM> for <NUM> minutes. Finally make sure homogeneous dispersion and complete defoamation.

A compound of Formula I, for example "ASO <NUM>" was formulated as a face cream for topical application to subjects. The face cream was prepared as described below. Given that there are lots of variations of topical cream possible, this preparation should be taken as an example and should not be interpreted to limit the scope of the current invention.

Claim 1:
A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof, for inducing exon skipping within human ACC2 (acetyl-CoA carboxylase <NUM>) pre-mRNA:
<CHM>
wherein,
n is an integer between <NUM> and <NUM>;
the compound of Formula I possesses at least a <NUM>-mer complementary overlap with the <NUM>-mer pre-mRNA sequence of [(<NUM>' → <NUM>') GGCCAUUUCGUCAGUAUC] in the human ACC2 pre-mRNA;
the compound of Formula I is fully complementary to exon/intron junction of the human ACC2 pre-mRNA;
S<NUM>, S<NUM>, •••, Sn-<NUM>, Sn, T<NUM>, T<NUM>, •••, Tn-<NUM>, and Tn are hydrido radical;
X and Y independently represent hydrido or substituted or non-substituted alkyloxycarbonyl radical;
Z represents substituted or non-substituted amino radical;
B<NUM>, B<NUM>, •••, Bn-<NUM>, and Bn are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and,
at least four of B<NUM>, B<NUM>, •••, Bn-<NUM>, and Bn are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV:
<CHM>
wherein,
R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> are hydrido radical;
L<NUM>, L<NUM> and L<NUM> are a covalent linker represented by Formula V covalently linking the basic amino group to the nucleobase moiety:
<CHM>
wherein,
Q<NUM> and Qm are substituted or non-substituted methylene (-CH<NUM>-) radical, and Qm is directly linked to the basic amino group;
Q<NUM>, Q<NUM>, •••, and Qm-<NUM> are independently selected from substituted or non-substituted methylene, and oxygen (-O-), radical; and
m is an integer between <NUM> and <NUM>.