Patent Description:
Alopecia is a disorder characterized by hair loss and hair thinning initially on the scalp. Androgenic alopecia, also referred to as "male pattern baldness," is caused by overt androgenic activity in hair follicles and surrounding tissue.

While androgenic alopecia affects both men and women, the disorder often shows up differently in men versus women. Males are likely to experience spot baldness, and females are more likely to experience overall hair thinning on the scalp. The prevalence of androgenic alopecia in males aged <NUM> to <NUM> is approximately <NUM>% [<NPL>)]. Androgenic alopecia is caused by changes in male steroid hormones known as androgens [<NPL>); <NPL>)].

Androgens regulate the release of sebum in sebaceous glands, the hair growth in hair follicles, libido systemically, and so on. Androgens stimulate the gradual transformation of small vellus follicles, making non-pigmented, fine, and short hairs in some areas to larger terminal follicles (e.g. face). In contrast to this androgen action on terminal follicles, however, gradual regression of terminal hair follicles to vellus follicles occurs on the temples and scalp vertex, which is often called as `androgen paradox'.

Androgenic Alopecia and DHT: 5α-reductase reduces testosterone into <NUM>α - dihydrotestosterone (DHT), an androgen more potent and effective than testosterone. A substantial increase in DHT production in frontal anagen hair follicles was observed in young balding males compared with non-balding males. [(<NPL>)] Males with androgenic alopecia tend to show a lower level of "total testosterone" than those without androgenic alopecia. Instead, the DHT level is higher in males with androgenic alopecia than in those without androgenic alopecia. DHT is produced from testosterone by 5α-reductase. Males with androgenic alopecia express a higher level of <NUM> a -reductase in hair follicles than those without androgenic alopecia. DHT is highly responsible for miniaturization of hair follicles, and therefore androgenic alopecia.

Finasteride and dutasteride inhibit 5α-reductase, and therefore decrease the DHT level available to androgen receptors in hair follicles and surrounding tissue. The two small molecule inhibitors have been used to treat male pattern baldness despite adverse effects originating from the down-regulation of systemic androgenic activity. The adverse effects include sexual dysfunction, dizziness, weakness, headache, runny nose, skin rash, and so on.

Topical AR Antagonist: Androgens express their pharmacologic activities by binding to androgen receptor (AR). AR antagonists bind to AR and inhibit the physiological function of androgens, and therefore may be used to treat androgenic alopecia if properly delivered to hair follicles and surrounding tissue. In order to avoid side effects incurred by the inhibition of systemic androgenic activity, AR antagonists are topically administered directly to scalp tissue.

Ketoconazole possesses weak AR antagonistic activity in addition to its famous antifungal activity. A shampoo containing <NUM>% ketoconazole (under a commercial brand name of Nizoral®) has been used to topically treat androgenic hair loss.

Topilutamide is an AR antagonist known as fludiril. Topilutamide is marketed as a <NUM>% topical formulation to treat androgenic alopecia in a number of European countries with a brand name of "Eucapil".

AR Protein or mRNA in Hair Follicles: In male and female subjects with androgenic alopecia, AR expression was found to be higher in frontal hair follicles than in occipital hair follicles. [<NPL>)] If AR expression is down-regulated by an agent selectively in hair follicles and surrounding tissue, such agent may safely treat androgenic alopecia without incurring adverse events caused by the systemic down-regulation of androgenic activity. In another literature, females with androgenic alopecia were found to show a higher level of AR mRNA in frontal and parietal hair follicles than in occipital hair follicles.

Ribosomal Protein Synthesis: Proteins are encoded by DNA (<NUM>-deoxyribose nucleic acid). In response to cellular stimulation, 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 cytosolic compartment. In the cytosol, 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>)].

An oligonucleotide binding to RNA in a sequence specific manner (i.e. complementarily) is called antisense oligonucleotide (ASO). ASO may tightly bind to an mRNA and inhibit the protein synthesis by ribosome along the mRNA in the cytosol. ASO needs to be present within cell in order to inhibit the ribosomal protein synthesis of its target protein.

Splicing Process: DNA is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. Pre-mRNA is then processed into mRNA following deletion of introns by a series of complex reactions collectively called "splicing" as schematically summarized in the diagram below. [<NPL>); <NPL>); <NPL>)].

Splicing is initiated by forming "splicesome E complex" (i.e. early splicesome complex) between pre-mRNA and splicing adapter factors. In "splicesome 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 splicesome complex. "Splicesome E complex" evolves into "splicesome A complex" following additional complexation with U2. The "splicesome A complex" undergoes a series of complex reactions to delete or splice out the intron to adjoin the neighboring exons.

Antisense Inhibition of Splicing: In the nucleus, ASO may tightly bind to a certain position within a pre-mRNA, and can interfere with the splicing process of the pre-mRNA into mRNA, producing the full-length mRNA or mRNA variant(s) lacking the target exon. Such mRNA(s) is called "splice variant(s)", encodes protein(s) smaller than the protein encoded by the full-length mRNA.

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

Unnatural Oligonucleotides: DNA or RNA oligonucleotide is susceptible to degradation by endogenous nucleases, limiting their therapeutic utility. To date, a large number of unnatural oligonucleotides have been developed and studied intensively. [<NPL>)] Some of them were found to show extended metabolic stability compared to DNA and RNA. Provided below are the chemical structures for a few number of representative unnatural oligonucleotides. Such oligonucleotide predictably binds to its 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 sulfur atom per monomer. Such a small structural change made PTO comparatively resistant to degradation by nucleases.

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

In order to increase PTO's in vitro cell membrane permeability, lipofection has been widely practiced. However, lipofection physically alters cell membrane, causes cytotoxicity, and therefore would not be safe for long term 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 membrane permeability. In order to overcome the poor membrane permeability, 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.

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 a certain population of 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>)] Like PTO, LNA also shows poor cell membrane permeability.

Phosphorodiamidate Morpholino Oligonucleotide: In phosphorodiamidate morpholino oligonucleotide (PMO), the backbone phosphate and <NUM>-deoxyribose of DNA are replaced with phosphoamidite 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(s) recognizing DNA or RNA. However, PMO doesn't readily penetrate cell membrane, either.

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 the prototype PNA are illustrated with the drawing provided below. Like DNA and RNA, PNA also selectively binds to complementary nucleic acid. [<NPL>)] 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 that 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 mammalian cell membrane.

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 above. Such modified nucleobases of cytosine, adenine, and guanine were found to predictably and complementarily hybridize with guanine, thymine, and cytosine, respectively. No. <CIT> (published as <CIT>); <CIT>; <CIT>]
<CHM>
<CHM>.

Incorporation of such modified nucleobases onto PNA resembles situations of lipofection. By lipofection, oligonucleotide molecules are wrapped with cationic lipid molecules such as lipofectamine, and such lipofectamine/oligonucleotide complexes tend to penetrate cell 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.

AR Antisense Oligonucleotide (AR ASO): In principle, an ASO targeting the AR mRNA can inhibit ribosomal protein synthesis of androgen receptor. There are reported cases of AR ASOs inhibiting AR expression in cells. For example, EZN-<NUM>, an LNA/DNA gapmer complementarily targeting the AR mRNA, down-regulated AR expression in tumor cells as well as in tumors of animal models for prostate cancer.

ASOs targeting either exon <NUM> or exon <NUM> of the AR mRNA inhibited AR expression in prostate cancer cells as well as in tumors of animal models for prostate cancer resistant to chemotherapy with Enzalutamide, an AR antagonist.

AR Down-regulation in Hair Follicles by Topical Application of AR ASO: Down-regulation of androgenic activity in hair follicles and surrounding tissue may be achieved by inhibiting AR expression in hair follicles and surrounding tissue. AR expression in hair follicles can be down-regulated with an AR ASO, if the ASO is delivered into hair follicles and surrounding tissue.

In order to avoid the side effects from the down-regulation of systemic androgenic activity, it is desired to have AR expression down-regulated locally in hair follicles and surrounding tissue for the treatment of androgenic alopecia. Topical application of an AR ASO to scalp skin would be the safest mode to inhibit AR expression locally in hair follicles and surrounding tissue, if the ASO is made or formulated to be readily delivered into hair follicles. To date, AR ASOs have been hardly used for topical treatment of androgenic alopecia. AR ASOs have been evaluated mostly for systemic administration to treat prostate cancer resistant to androgen ablation therapy.

The present invention provides a peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof:
<CHM>
wherein,.

The compound of Formula I induces alternative splicing of the human AR pre-mRNA, yields AR mRNA splice variant(s) lacking "exon <NUM>", and therefore is useful to safely treat dermatological indications or conditions involving androgenic activity upon topical administration.

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

The compound of Formula I tightly binds to the <NUM>' splice site of "exon <NUM>" of the human AR pre-mRNA transcribed from the human AR gene. [NCBI Reference Sequence: NC_000023. <NUM>] The <NUM>-mer AR pre-mRNA sequence consisting of a <NUM>-mer from "exon <NUM>" and a <NUM>-mer from "intron <NUM>" unequivocally reads [(<NUM>' —> <NUM>') GUGGGCCAAGGCCUUGCCUG-GUAAGGAAAAGGGAAGUGGG], although the exon and intron number may vary depending on AR mRNA transcript. The <NUM>-mer pre-mRNA sequence may be alternatively denoted as [(<NUM>' ----+ <NUM>') GUGGGCCAAGGCCUUGCCUG | guaaggaaaagggaaguggg], wherein the exon and intron sequences are expressed with "capital" and "small" letters, respectively, and the exon/intron junction is expressed with " | ".

The <NUM>-mer pre-mRNA sequence of [(<NUM>' —> <NUM>') CCUUGCCUGGUAAGGAA] adopted to describe the compound of Formula I in this invention consists of <NUM>-mer in the AR "exon <NUM>" and <NUM>-mer in the AR "intron <NUM>". Thus the <NUM>-mer pre-mRNA sequence may alternatively read [(<NUM>' —> <NUM>') CCUUGCCUG | guaaggaa].

The compound of Formula I tightly binds to the target <NUM>' splice site of exon <NUM> in the human AR pre-mRNA, and interferes with the formation of "splicesome early complex" involving the compound's target exon. Since the compound of this invention sterically inhibits the formation of "splicesome early complex", the AR "exon <NUM>" is spliced out to yield an AR mRNA splice variant or variants lacking "exon <NUM>". Consequently the compound of this invention induces the skipping of "exon <NUM>".

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

Owing to the high binding affinity, the PNA derivative of this invention potently induces the skipping of "exon <NUM>" in cells even with a complementary overlap of as small as <NUM>-mer with the <NUM>' splice site of "exon <NUM>", although such a small number of overlap may increase the risk of cross reactivity with other pre-mRNAs. If the PNA derivative of this invention is used for topical therapeutic purposes, the risk of the cross reactivity is predicted to be considerably attenuated.

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 (i.e. non-naturally 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. A skilled person in the field may easily figure out that variations of unnatural nucleobases are possible for specific positions in the PNA compound of Formula I as long as such variations meet the desired complementarity with its target pre-mRNA sequence.

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 alkylacyl 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 an oligonucleotide to the target pre-mRNA sequence over substituents in the N-terminus or C-terminus.

The compound of Formula I possesses good cell permeability and can be readily delivered into cell if treated as "naked" oligonucleotide as exemplified in the prior art [<CIT>, published as <CIT>]. Thus the compound of this invention induces the skipping of "exon <NUM>" in the human AR pre-mRNA to yield AR mRNA splice variant(s) lacking AR "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 the AR "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 the AR "exon <NUM>" in target skin. The compound of Formula I does not require a formulation to increase trans-dermal delivery for a topical therapeutic or biological activity. Usually the compound of Formula I is dissolved in water and co-solvent, and topically or trans-dermally administered at sub-picomolar concentration to elicit the desired therapeutic or biological activity in the target skin. The compound of this invention does not need to be heavily or invasively formulated to elicit the topical therapeutic activity.

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.

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.

Of highest 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, 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), A(pOq), G(p), and G(pOq). As discussed in the prior art [<CIT>, published as <CIT>], C(pOq) is regarded as a "modified cytosine" PNA monomer due to its hybridization for "guanine". A(p) and A(pOq) are taken as "modified adenine" PNA monomers for their hybridization for "thymine". Likewise G(p) and G(pOq) are considered to be "modified guanine" PNA monomers for their base pairing with "cytosine".

<FIG> unequivocally illustrates the chemical structures for a variety of abbreviations for substituents used for diversifying the N-terminus or C-terminus of the PNA derivative of Formula I in this invention.

In order to illustrate the abbreviations for the PNA derivatives, the chemical structure for the PNA derivative abbreviated as "(N —> C) Fethoc-GA(<NUM>)A-GC(1O2)C-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-NH<NUM>" is provided in <FIG>. As another illustration, the chemical structure for the PNA derivative abbreviated as "(N ----+ C) Benzoyl-Lys-Val-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-NH<NUM>" is provided in <FIG>.

The <NUM>-mer PNA sequence of "(N —> C) Fethoc-GA(<NUM>)A-GC(1O2)C-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-NH<NUM>" is equivalent to the DNA sequence of "(<NUM>' —> <NUM>') GAA-GCC-AGG-CAA-G" in binding to its complementary binding with pre-mRNA. The <NUM>-mer PNA has a <NUM>-mer complementary overlap with the <NUM>-mer sequence marked as "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' —> <NUM>') GCCUUGCCUG | g"uaag"gaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA. It is noted that the four single mismatches in "intron <NUM>" is marked as "uaag".

The <NUM>-mer PNA sequence of "(N —> C) Benzoyl-Lys-Val-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-NH<NUM>" is equivalent to the DNA sequence of "(<NUM>' —> <NUM>') CTT-ACC-AGG-CAA-G", which has a <NUM>-mer complementary overlap with the <NUM>-mer sequence marked as "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' —> <NUM>') GCCUUGCCUG | guaaggaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA.

The <NUM>-mer PNA sequence of "(N ----+ C) Ac-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG-C(1O2)TA(<NUM>)-G-NH<NUM>" is equivalent to the DNA sequence of "(<NUM>' —> <NUM>') CTT-ACC-AGG-CTA-G", which has a <NUM>-mer complementary overlap with the <NUM>-mer sequence as marked "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' → <NUM>') GCCU"U"GCCUG | guaaggaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA. It is noted that the single mismatch in exon <NUM> is marked as "U".

The <NUM>-mer PNA sequence of "(N ----+ C) Fethoc-TTT-TCC(1O2)-TTA(<NUM>)-CCA(<NUM>)-GG(<NUM>)C-A(<NUM>)A-NH<NUM>" is equivalent to the DNA sequence of "(<NUM>' ----+ <NUM>') TTT-TCC-TTA-CCA-GGC-AA", which has a <NUM>-mer complementary overlap with the <NUM>-mer sequence marked as "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' —> <NUM>') GCCUUGCCUG | guaaggaaaa ] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA.

The present invention provides a PNA derivative of Formula I which is selected from the group of specifically preferred compounds enlisted below, or a pharmaceutically acceptable salt thereof:.

Also provided are a peptide nucleic acid derivative selected from the group consisting of: (N → C) Fethoc-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-NH<NUM>, (N → C) Fethoc-TC(1O2)C-TTA(<NUM>)-CCA(<NUM>)-GGC(1O2)-AA(<NUM>)G-G(<NUM>)-NH<NUM>, (N → C) Fethoc-TC(1O2)C-TTA(<NUM>)-CCA(<NUM>)-GGC(1O2)-AA(<NUM>)G-G(<NUM>)-NH<NUM>, (N → C) Fethoc-TA(<NUM>)C-CAG(<NUM>)-GC(1O2)A-A(<NUM>)GG(<NUM>)-C-NH<NUM>, (N → C) Fethoc-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG(<NUM>)-CA(<NUM>)A-NH<NUM>, (N → C) Fethoc-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG(<NUM>)-CA(<NUM>)A-NH<NUM>, (N → C) Ac-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG(<NUM>)-CA(<NUM>)A-NH<NUM>, or (N → C) Fethoc-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG(<NUM>)-CA(<NUM>)A-NH<NUM>, 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. 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>, published as <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 (watre/acetonitrile or water/methanol with <NUM>% TFA) and characterized by mass spectrometry.

Scheme <NUM> illustrates a typical monomer elongation cycle adopted in the SPPS of this invention, and procedural details are provided below. To a skilled person in the field, however, lots of minor variations are 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. <CHM>
<CHM>.

[Activation of H-Rink-ChemMatrix Resin] <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 either with an Fmoc-PNA monomer or with an Fmoc-protected amino acid derivative.

[DeFmoc] The resin was vortexed in <NUM> <NUM>% piperidine/DMF 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> mLMC.

[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" under basic coupling conditions. The chemical structure of "Fethoc-OSu" [<NPL>, C<NUM>H<NUM>NO<NUM>, MW <NUM>] 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 residue was triturated with diethylether, 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> are exemplary HPLC chromatograms for "ASO <NUM>" before and after HPLC purification, respectively. The oligomer sequence of "ASO <NUM>" is as provided in Table <NUM>.

PNA derivatives of this invention were prepared according to the synthetic procedures provided above or with minor modifications. Table <NUM> provides examples of AR ASOs of the present invention along with structural characterization data by mass spectrometry. Provision of the AR ASOs 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.

The PNA derivatives in Table <NUM> 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 in Table <NUM> and fully complementary DNAs show Tm values too high to be reliably determined in aqueous buffer solution, since the buffer solution tends to boil off during the Tm measurement.

Tm values were determined on an 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 minute and slowly cooled down to ambient temperature over several minutes. Then the solution was transferred into a <NUM> quartz UV cuvette equipped with an air-tight cap, and subjected to a Tm measurement at <NUM> on an Agilent <NUM> UV/Visible spectrophotometer or a similar one as described in the prior art [<CIT>, published as <CIT>] or with minor modifications. The <NUM>-mer complementary 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, and 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 within the PNA marked as "bold" and "underlined" in [(N → C) Fethoc-C(1O2)TT-A(<NUM>)CC-A(<NUM>)GG-C(1O2)AA(<NUM>)-G-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 within the PNA marked as "bold" and "underlined" in [(N → C) Fethoc-C(<NUM>)TT-A(<NUM>)CC-A(<NUM>)GG-C(<NUM>)AA(<NUM>)-G-NH<NUM>].

PNA derivatives of Formula I were evaluated for their biological activities in vitro and in vivo. The biological examples provided below are provided as examples to illustrate the biological profiles of such PNA derivatives of Formula I, and therefore should not be interpreted to limit the scope of the current invention.

"ASO <NUM>" specified in Table <NUM> is a <NUM>-mer antisense oligonucleotide which has a <NUM>-mer full complementary overlap with the <NUM>-mer sequence marked as "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' —> <NUM>') GCCUUGCCUG | guaaggaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA.

"ASO <NUM>" was evaluated by AR nested PCR for its ability to induce the skipping of AR "exon <NUM>" in MCF7 cells (Cat. Number: HTB-<NUM>, ATCC). The employed procedures are detailed as follows.

[Cell Culture & ASO Treatment] MCF7 cells were grown in EMEM medium supplemented with <NUM>% FBS, <NUM>% streptomycin/penicillin, and <NUM>/ml bovine insulin under <NUM>% CO<NUM> atmosphere at <NUM>. Cells were sub-cultured in <NUM> culture dish prior to treatment with "ASO <NUM>" at <NUM> aM to <NUM> fM.

[RNA Extraction] MCF7 cells were incubated with or without "ASO <NUM>" for <NUM> hours. Total RNA was extracted from cells in <NUM> culture dish using "Universal RNA Extraction Kit" (Cat. No. <NUM>, Takara) according to the manufacturer's instructions.

[cDNA Synthesis by One-step PCR] <NUM> ng of RNA template was used in a <NUM>µL reverse transcription reaction using Super Script® One-Step RT-PCR kit with platinum® Taq polymerase (Cat. No. <NUM>-<NUM>, Invitrogen) against a set of gene-specific primers [exon 3_forward: (<NUM>' → <NUM>') TGGGTGTCACTATGGAGC, and exon 9_reverse: (<NUM>' → <NUM>') GGGTGT-GGAAATAGATGGG] according to the following cycle conditions: <NUM> for <NUM> and <NUM> for <NUM>, followed by <NUM> cycles of <NUM> sec at <NUM>, <NUM> sec at <NUM>, and <NUM> at <NUM>.

[Nested PCR Amplification] Throughout the amplification process, was used a unique amplification technique (touch up as increasing annealing temperature per cycle) that worked efficiently and specifically over a temperature range, rather than at one specific annealing temperature (i.e. conventional PCR method). <NUM>µL of cDNA was further amplified in a <NUM>µL nested PCR (Invitrogen) reaction against a set of primers [exon 3_forward: (<NUM>' →.

<NUM>') TGGGTG-TCACTATGGAGC, and exon 7n_reverse: (<NUM>' → <NUM>') GGGGTGATTTGGAGCCAT] according to the following cycle conditions: initial <NUM> cycles [<NUM> for <NUM> sec, <NUM> for <NUM> sec (+<NUM> every cycle), <NUM> for <NUM> sec], followed by <NUM> cycles [<NUM> for <NUM> sec, <NUM> for <NUM> sec, and <NUM> for <NUM> sec].

[Identification of Exon Skipping Products] The PCR products were subjected to electrophoretic separation on a <NUM>% agarose gel. The bands of target size were collected and analyzed by Sanger Sequencing. In <FIG>, there were three treatment-related PCR product bands assignable to AR mRNA splice variants lacking "exon <NUM>". "ASO <NUM>" was found to induce the skipping of "exon <NUM>", "exons <NUM>-<NUM>", and "exons <NUM>-<NUM>", although the ratio of the skipping products appeared to be dependent on the ASO concentration. <FIG> provides the actual sequencing data for the skipping band of "exons <NUM>-<NUM>" in <FIG> as an example for Sanger Sequencing.

"ASO <NUM>" was evaluated for its ability to down-regulate the human AR mRNA by qPCR with SYBR Green detection.

MCF7 cells were sub-cultured in <NUM> culture medium in <NUM> culture dish, and treated or with "ASO <NUM>" at <NUM> zM (negative control) to <NUM> aM (<NUM> culture dishes per each concentration). <NUM> hours later, total RNA was extracted with "MiniBEST Universal RNA Extraction Kit" according to the manufacturer's instructions (Cat. No. <NUM>, Takara). <NUM> ng of RNA template were used to synthesize cDNA for a <NUM>µL reverse transcription reaction using Oligo-dT according to the manufacturer's instructions (Cat. No. 6110A, Takara). cDNA was then subjected to the <NUM>st PCR against a set of primers covering "exon <NUM>" to "exon <NUM>" [Exon 3_forward: (<NUM>' → <NUM>') TGGGTGTCACTATGGAGC, and Exon 9_reverse: (<NUM>' → <NUM>') GGGTG-TGGAAATAGAT-GGG] according to the following cycle conditions: <NUM> for <NUM> followed by <NUM> cycles of <NUM> sec at <NUM>, <NUM> sec at <NUM>, and <NUM> at <NUM>.

The <NUM>st PCR products were diluted by <NUM>,<NUM> times, and <NUM>µL of each diluted PCR product was subjected to a <NUM>µL Real-Time PCR reaction against sets of exon specific primers sets [Exon 4_forward(q): (<NUM>' → <NUM>') GACCATGTTTTGCCCATTG and Exon 4_reverse(q): (<NUM>' → <NUM>') GGCTCTTTTGAAGAAGACC for exon <NUM>; Exon 5_forward(q): (<NUM>' → <NUM>') GAAACAGAAGTA-CCTGTGC and Exon 5_reverse(q): (<NUM>' → <NUM>') GTCATCCCTGCTTCATAAC for exon <NUM>; and Exon 6_forward(q): (<NUM>' → <NUM>') CGGAAGCTGAAGAAACTTG and Exon 6_reverse(q): (<NUM>' → <NUM>') CACTTGACCACGTGTACAAG for exon <NUM>]. The PCR reactions were monitored by SYBR Green (Takara, Japan). Cycle Conditions: <NUM> for <NUM> followed by <NUM> cycles <NUM> sec at <NUM>, and <NUM> sec at <NUM>.

<FIG> provides the qPCR data obtained therefrom. The relative expression level of exons <NUM>-<NUM> significantly decreased as the ASO concentration was increased from <NUM> zM to <NUM> zM. At <NUM> zM, the exon message levels decreased by ca <NUM> to <NUM>%. At <NUM> aM, however, the exon message levels rebounded to near the levels of the negative control (no ASO treatment). The strange dose response pattern of the qPCR data could be due to a transcription upregulation by the "exon intron circular RNA (EIciRNA)" accumulated during the exon skipping with "ASO <NUM>".

Although "ASO <NUM>" specified in Table <NUM> is a <NUM>-mer antisense oligonucleotide originally designed to complementarily target the junction of "exon <NUM>" and "exon <NUM>" within the human AR mRNA. "ASO <NUM>" has a <NUM>-mer complementary overlap with the <NUM>-mer sequence as marked "bold" and "underlined" in the <NUM>-mer RNA sequence [(<NUM>' → <NUM>') GCCUUGCCUG | g"uaag"gaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" within the human AR pre-mRNA. It is noted that the four single mismatches in intron <NUM> are marked as "uaag". Thus "ASO <NUM>" may be regarded as an antisense oligonucleotide targeting the human AR pre-mRNA, although only with a <NUM>-mer complementary overlap out of the <NUM>-mer sequence.

"ASO <NUM>" was evaluated for its ability to down-regulate the human AR mRNA by qPCR according to the protocol described in "Example <NUM>".

<FIG> provides the qPCR data obtained therefrom. The relative expression level of "exons <NUM>-<NUM>" significantly decreased as the ASO concentration was increased from <NUM> zM to <NUM> zM. At <NUM> zM, the exon message levels decreased by more than <NUM>%. At <NUM> aM, however, the exon message levels rebounded to ca <NUM>% of the negative control (no ASO treatment). The strange dose response pattern of the qPCR data could be due to a transcription upregulation by the "exon intron circular RNA (EIciRNA)" accumulated during the exon skipping with "ASO <NUM>".

"ASO <NUM>" specified in Table <NUM> is a <NUM>-mer antisense oligonucleotide which has a <NUM>-mer full complementary overlap with the <NUM>-mer sequence as marked "bold" and "underlined" in the <NUM>-mer pre-mRNA sequence [(<NUM>' → <NUM>') GCCUUGCCUG | guaaggaaaa] spanning the junction of "exon <NUM>" and "intron <NUM>" of the human AR pre-mRNA.

"ASO <NUM>" was evaluated for its ability to down-regulate the human AR mRNA (full-length) by qPCR according to the protocol described in "Example <NUM>".

<FIG> provides the qPCR data obtained therefrom. The relative expression level of "exons <NUM>-<NUM>" significantly decreased by <NUM> ~ <NUM>% in MCF7 cells treated with "ASO <NUM>" at <NUM> zM to <NUM>,<NUM> zM.

MCF7 cells were sub-cultured in <NUM> culture dish containing <NUM> culture medium, and treated with "ASO <NUM>" at <NUM> zM (negative control), or <NUM> zM to <NUM> aM. <NUM> culture dishes were used for <NUM> negative controls. <NUM> hours later, cells were washed <NUM> times with cold PBS, and then subjected to lysis with <NUM>µL 1X cell lysis buffer (Cat. No. <NUM>, Cell Signaling Tech) supplemented with 1X protease inhibitor (Cat. No. P8340, Sigma). The lysates were collected in <NUM> e-tube. <NUM>µL of each lysate was mixed with <NUM>µL 3X sample buffer, and boiled for <NUM> at <NUM>. <NUM>µL of each lysate (<NUM> lysates in total). <NUM> negative controls and <NUM> ASO treatment samples) was subjected to electrophoretic separation on a <NUM>% SDS-PAGE gel, and transferred onto a <NUM> PVDF membrane. The membrane was probed with anti-AR antibody (Cat. No. <NUM>, Cell Signaling Tech) and anti-β-actin antibody (Cat. No. sc4778, Santa Cruz). <FIG> provides the AR Western blot data obtained therefrom. Multiple (negative) control samples were used to overcome technical artifacts of western blot procedures. Except for the AR band for (negative) "control <NUM>", the AR band intensity of the lysates with ASO treatment was considerably weaker than that of the lysates without ASO treatment, which unequivocally indicates that "ASO <NUM>" inhibits the expression of the full-length AR protein in MCF7 cells.

"ASO <NUM>" was evaluated for its ability to inhibit AR protein expression at <NUM> zM to <NUM> aM in MCF7 cells according to the procedures described in "Example <NUM>".

<FIG> provides the AR Western blot data obtained with MCF7 cells treated with "ASO <NUM>" at <NUM> zM (negative control, no ASO treatment), or <NUM> zM to <NUM> aM. Multiple (negative) control samples were used to overcome technical artifacts of western blot procedures. The AR band intensity of the lysates with ASO treatment was considerably weaker than that of the neighboring lysates without ASO treatment, which unequivocally indicates that "ASO <NUM>" inhibits the expression of the full-length AR protein in MCF7 cells.

"ASO <NUM>" was evaluated for its ability to promote hair growth in C57BL/<NUM> mice upon topical administration as follows. The target sequence of "ASO <NUM>" in the human AR pre-mRNA is conserved in the mouse AR pre-mRNA. Thus the in vivo therapeutic findings in mice may be extrapolated to human cases without much ambiguity.

[Hair Removal and Grouping] In Day <NUM>, <NUM> week old female C57BL/<NUM> mice were anesthetized with zoletil/rompun, and the hair in the back was cut and removed with a clipper and wax, respectively. Mice with flawless (i.e. spotless) hair removal were selected and randomly assigned into three groups (<NUM> animals per group).

[Topical Administration] The topical solutions of "ASO <NUM>" were prepared by diluting a mother stock solution of "ASO <NUM>" to <NUM> fM or <NUM> fM in aqueous <NUM>%(v/v) ethanol supplemented with <NUM>%(v/v) glycerin. About <NUM>µL of <NUM> (negative control), <NUM>, or <NUM> fM "ASO <NUM>" was topically administered in the back of each animal using a cotton ball in Days <NUM>, <NUM>, <NUM>, and <NUM>.

[Digital Image Scoring for Hair Growth] For scoring the hair growth, the animals were anesthetized and photographed by group as shown in <FIG> using a digital camera at a fixed value of exposure time and illumination. The digital image for the area of the hair removal for each animal was selected and digitally scored for the average brightness over the selected area using "ImageJ" program. Lower brightness score is taken as faster hair growth. Brightness scores of individual animals were combined by group, and subjected to statistical analysis by student's t-test. <FIG> summarizes the relative brightness scores of the ASO treated groups against the control group. The relative brightness score tended to decrease with days in the treatment groups. In Day <NUM>, the <NUM> fM group was significantly lower in the brightness score than the non-treated group. Thus, "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> fM.

[Scoring by Hair Weight] In Day <NUM>, the animals were anesthetized and the hair in the back was cut with a clipper. The hair samples from individual animals were combined by group, and weighed to evaluate the hair growth between Day <NUM> and Day <NUM>. The average hair weight was <NUM>/animal for the control group, <NUM>/animal for the <NUM> fM treatment group, and <NUM>/animal for the <NUM> fM treatment group. Thus "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> fM as well as <NUM> fM.

[AR IHC of Skin Sample] Following the shaving in Day <NUM>, the mice received a single topical administration of either vehicle or "ASO <NUM>" according to the group. In Day <NUM>, the skin of the area with hair removal was sampled for immunohistochemistry (IHC) analysis against androgen receptor. The skin samples were cryo-sectioned and subjected to immunostaining in series with a primary anti-AR antibody (Cat. No. sc-<NUM>, Santa Cruz) at <NUM>:<NUM> dilution, with a secondary anti-IgG (Cat No. BA-<NUM>, Vector) at <NUM>:<NUM> dilution, and then with Dylight <NUM>-steptavidin (Cat No. SA-<NUM>, Vector, CA, USA) at <NUM>:<NUM> dilution for red fluoresence tagging. The IHC images were captured on an Olympus fluorescence microscope for changes in the AR expression level upon topical treatment with "ASO <NUM>".

<FIG> is a representative set of AR IHC images demonstrating that the AR expression in hair follicles was markedly inhibited in hair follicles upon the topical administrations of "ASO <NUM>" at <NUM> fM or <NUM> fM. The DAPI staining images were provided to locate the hair follicles in the IHC images. It is interesting to note that AR expression decreased even in the muscle layer underneath the dermis upon the topical administrations of "ASO <NUM>" at <NUM> fM or <NUM> fM. Thus "ASO <NUM>" is readily delivered into the dermis as well as the muscle layer underneath the dermis upon topical administration, and potently inhibits the expression of AR.

"ASO <NUM>" was evaluated for its ability to promote hair growth in C57BL/<NUM> mice upon topical administration as detailed below. The target sequence of "ASO <NUM>" in the human AR pre-mRNA is conserved in the mouse AR pre-mRNA. Thus the in vivo therapeutic findings in mice may be extrapolated to human cases without much ambiguity.

[Topical Administration] The topical solutions of "ASO <NUM>" were prepared by diluting a mother stock solution of "ASO <NUM>" to <NUM>, <NUM>, or <NUM> fM in aqueous <NUM>%(v/v) ethanol supplemented with <NUM>%(v/v) glycerin. About <NUM>µL of each ASO solution or vehicle (negative control) was topically administered to in the back of an animal using a cotton ball in Days <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

[Digital Image Scoring for Hair Growth] <FIG> summarizes the relative brightness scores of the ASO treated groups against the negative control group. The relative brightness score tended to decrease with days in the ASO treatment groups. In Day <NUM>, the treatment groups of <NUM> fM and <NUM> fM were significantly lower in the brightness score than the non-treated group. Thus, "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> to <NUM> fM.

[Scoring by Hair Weight] In Day <NUM>, the animals were anesthetized and the hair in the back was cut and collected. The hair samples from individual animals were combined by group, and weighed to evaluate the hair growth between Day <NUM> and Day <NUM>. The treatment groups yielded marked increases in the hair weight compared to the control group. The average hair weights of the <NUM> fM, <NUM> fM, and <NUM> fM groups were <NUM>%, <NUM>%, and <NUM>% of the negative control group, respectively. Thus "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> to <NUM> fM.

Following the shaving in Day <NUM>, the animals received a single topical administration of either vehicle or "ASO <NUM>" at <NUM> fM, <NUM> fM, or <NUM> fM. In Day <NUM>, the hair in the back was collected by shaving with a clipper to determine the total amount of hair growth between Days <NUM> and <NUM>. The average hair weights of the <NUM> fM, <NUM> fM, and <NUM> fM groups were <NUM>,<NUM>%, <NUM>,<NUM>%, and <NUM>% of the non-treated group, respectively. Thus "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> to <NUM> fM.

[Topical Administration] The topical solutions of "ASO <NUM>" were prepared by diluting a mother stock solution of "ASO <NUM>" to <NUM>, <NUM>, or <NUM> fM in aqueous <NUM>%(v/v) ethanol supplemented with <NUM>%(v/v) glycerin. About <NUM>µL of each ASO solution or vehicle (negative control) was topically administered to in the back of an animal using a cotton ball in Day <NUM>.

[Digital Image Scoring for Hair Growth] <FIG> summarizes the relative brightness scores of the ASO treated groups against the control group. The relative brightness score tended to decrease with days in the treatment groups. In Day <NUM>, the treatment groups of <NUM> fM and <NUM> fM were significantly lower in the brightness score than the non-treatment group. Thus, "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> to <NUM> fM.

[Scoring by Hair Weight] In Day <NUM>, the animals were anesthetized and the hair in the back was cut and collected. The hair samples from individual animals were combined by group, and weighed to evaluate the hair growth between Day <NUM> and Day <NUM>. The treatment groups yielded modest increases in the hair weight compared to the control group. The average hair weights of the <NUM> fM, <NUM> fM, and <NUM> fM groups were <NUM>%, <NUM>%, and <NUM>% of the non-treated group, respectively. Thus "ASO <NUM>" was concluded to promote hair growth upon topical administrations at <NUM> to <NUM> fM.

[AR Immunohistochemistry of Skin Samples] After the hair cut in Day <NUM>, The animals topically received further either vehicle or "ASO <NUM>" at <NUM>, <NUM>, or <NUM> fM in Days <NUM> and <NUM>. Skin samples of the back were collected in Day <NUM> for immunohistochemical analysis against androgen receptor. The skin samples were subjected to immunostaining against androgen receptor as described in "Example <NUM>". IHC images were captured on a Zeiss slide scanner. <FIG> is a representative set of AR IHC images. It is interesting to note that the number of hair follicles increased markedly in the ASO treatment groups compared to the negative control group. It would be difficult to clearly state that the AR expression in hair follicles decreased in the treatment groups due to the marked increases in the number of hair follicles in the treatment groups. Nevertheless, the AR expression in the muscle layer underneath the dermis markedly decreased in the treatment groups compared to the control group. The most notable decrease was observed with the <NUM> fM treatment group. Taken together the IHC findings in the animals treated with "ASO <NUM>", "ASO <NUM>" promotes hair growth by inhibiting AR expression to increase the number of hair follicles at all the tested doses, most notably at <NUM> fM.

"ASO <NUM>" was evaluated for its ability to down-regulate the human AR mRNA by qPCR adopting a TaqMan probe.

MCF7 cells sub-cultured in <NUM> culture medium in <NUM> culture dish, and treated or with "ASO <NUM>" at <NUM> zM (negative control) to <NUM> aM (<NUM> culture dishes per each concentration). <NUM> hours later, total RNA was extracted by "MiniBEST Universal RNA Extraction Kit" according to the manufacturer's instructions (Cat. No. <NUM>, Takara).

<NUM> ng of RNA template were used to synthesize cDNA for a <NUM>µL reverse transcription reaction using One-Step RT-PCR kit (Invitrogen) against a set of exon specific primers of [exon 3_forward: (<NUM>' —> <NUM>') TGGGTGTCACTATGGAGC ; and exon 9_reverse: (<NUM>' → <NUM>') GGGTGT- GGAAATAGATGGG] according to the following cycle conditions: <NUM> for <NUM> and <NUM> for <NUM>, followed by <NUM> cycles of <NUM> sec at <NUM>, <NUM> sec at <NUM>, and <NUM> at <NUM>.

The cDNA solutions were diluted by <NUM> times, and <NUM>µL of each diluted PCR product was subjected to a <NUM>µL Real-Time PCR reaction against a set of exon specific primers of [exon 4_forward: (<NUM>' —> <NUM>') TTGTCCATCTTGTCGTCTT; and exon 5_reverse: (<NUM>' —> <NUM>') CCTCTC-CTTCCTCCTGTA] according to the following cycle conditions: <NUM> for <NUM> followed by <NUM> cycles <NUM> sec at <NUM>, and <NUM> sec at <NUM>. The qPCR reaction was monitored with a TaqMan probe of [(<NUM>' ----+ <NUM>') TTTCTTCAG-ZEN-CTTCCGGGCTC-3IABkFQ].

<FIG> provides the qPCR data obtained therefrom. The relative expression level of exons <NUM>-<NUM> significantly decreased by ca <NUM> to <NUM>% in MCF7 cells treated with "ASO <NUM>" at <NUM> zM to <NUM> aM.

"ASO <NUM>" was evaluated for its ability to inhibit AR expression in mice as follows. <NUM> weeks old male C57BL/<NUM> mice were randomly assigned to <NUM> groups of negative control (no ASO treatment), <NUM> pmole/Kg "ASO <NUM>" and <NUM> pmole/Kg "ASO <NUM>". (<NUM> animals per group) Mice subcutaneously received either vehicle or ASO dissolved in vehicle (PBS) 2X per week for <NUM> weeks according to the dosing group. Three days post the final dosing, the animals were anesthetized with zoletil/rompun and subjected to tissue or organ sampling for AR IHC by paraffin blolck.

The AR protein was probed in series with a primary antibody (Cat. No. sc-<NUM>, Santa Cruz) at <NUM>: <NUM> dilution, a secondary anti-rabbit IgG (Cat. No. BA-<NUM>, VECTOR) at <NUM>:<NUM> dilution, and Dylight <NUM>-Streptavidin (Cat. No. SA-<NUM>, VECTOR) at <NUM>:<NUM>. Nucleus was stained with DAPI. IHC fluorescence images were captured on a Zeiss slide scanner.

Claim 1:
A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof:
<CHM>
wherein,
n is an integer between <NUM> and <NUM>;
the compound of Formula I possesses at least a <NUM>-mer complementary overlap with a <NUM>-mer RNA sequence of [(<NUM>' —> <NUM>') CCUUGCCUGGUAAGGAA] within the human androgen receptor pre-mRNA;
S<NUM>, S<NUM>, ..., Sn-<NUM>, Sn, T<NUM>, T<NUM>, ..., Tn-<NUM>, and Tn independently represent deuterido, hydrido, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical;
X and Y independently represent hydrido [H], formyl [H-C(=O)-], aminocarbonyl [NH<NUM>-C(=O)-], substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, substituted or non-substituted aryloxycarbonyl, substituted or non-substituted alkylaminocarbonyl, substituted or non-substituted arylaminocarbonyl, substituted or non-substituted alkylsulfonyl, or substituted or non-substituted arylsulfonyl radical;
Z represents hydrido, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, or substituted or non-substituted aryl 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 with a substituted or non-substituted amino radical covalently linked to the nucleobase moiety.