RU2778786C9 - Tyrosinase antisense oligonucleotides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a derivative of peptide nucleic acid, a pharmaceutical composition for the treatment of diseases or conditions related to the expression of a human tyrosinase gene, including effective amount of the derivative of peptide nucleic acid, a pharmaceutical composition for the treatment of hyperpigmentation related to the expression of the human tyrosinase gene, including effective amount of the derivative of peptide nucleic acid, a method for the treatment of diseases or conditions related to the expression of the human tyrosinase gene, including administration of the derivative of peptide nucleic acid, a method for the treatment of hyperpigmentation related to the expression of the human tyrosinase gene, including the administration of the derivative of peptide nucleic acid, the use of the derivative of peptide nucleic acid in the production of a drug for the treatment of diseases or conditions related to the expression of the human tyrosinase gene, and the use of the derivative of peptide nucleic acid in the production of a drug for the treatment of hyperpigmentation related to the expression of the human tyrosinase gene.

EFFECT: invention expands the arsenal of remedies complementary purposefully affecting pre-mRNA of human tyrosinase.

9 cl, 22 dwg, 3 tbl, 6 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to peptide nucleic acid derivatives that complementarily target human tyrosinase pre-mRNA to improve tyrosinase-mediated skin pigmentation.

BACKGROUND OF THE INVENTION

Melanin generally refers to dark natural and polymeric pigments produced in living organisms. There are three types of melanin i.e. eumelanin, pheomelanin and neuromelanin. Eumelanin and pheomelanin are synthesized in the skin. Simultaneously, neuromelanin is melanin found in the brain, and its physiological functions have not yet been elucidated.

Melanin in the skin effectively absorbs UV (ultraviolet) radiation and protects animals from UV (ultraviolet) radiation in natural sunlight. Thus, tanning is a natural process by which animals protect themselves from UV exposure in the sun.

The amino acid tyrosine polymerizes to eumelanin after a series of complex reactions in the presence of tyrosinase. Eumelanin refers to polymers of 5,6-dihydroxyindole (dihydroxyindole, DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6-dihydroxyindole-2-carboxylic acid, DHICA). Pheomelanin is formed by the polymerization of tyrosine by tyrosinase in the presence of cysteine or glutathione.

Scheme 1 summarizes reactions involving TYR (tyrosinase), TYRP-1 (tyrosinase-dependent protein 1, i.e. DHICA oxidase) and TYRP-2 (tyrosinase-dependent protein 2, i.e. DOPAchrome (dopachrome) tautomerase) during the biosynthesis of eumelanin and pheomelanin. [Int. J. Mol. sci. vol 10,4066-4087 (2009)].

Scheme 1

Given that tyrosinase (TYR) plays a central role in melanin biosynthesis in the skin, TYR inhibitors have been used or developed as cosmetic ingredients to reduce excessive skin pigmentation, i.e. hyperpigmentation. Several publications [Int. J. Cosmetic Sci. vol 33, 210-221 (2011); Int. J. Mol. sci. vol 10, 2440-2475 (2009)] TYR inhibitors have been tested for their effects on melanogenesis.

Hydroquinone (1,4-dihydroxybenzene) has been used for the topical treatment of hyperpigmentation for decades. [JAMA vol 194(9), 965-967 (1965)] Hydroquinone is known to inhibit TYR activity by binding to the active site of tyrosinase. In 2006, the US FDA withdrew previous registrations for all over-the-counter (OTC) products containing hydroquinone due to safety concerns, including potential carcinogenicity. Hydroquinone induces reactive oxygen species (ROS) by damaging membrane lipids and proteins, including tyrosinase. Oxidative damage to membrane lipids irreversibly deprives melanocytes.

Arbutin (hydroquinone-O-β-D-glucopyranoside) is a glycosylated hydroquinone extracted from the bearberry plant. Arbutin is also found in wheat husks and pear skins. Like hydroquinone, arbutin inhibits melanogenesis by competitive binding to the TYR active site. [J. Pharmacol. Exp. Ther. vol 276(2), 765-769 (1996)]. Arbutin is less cytotoxic to melanocytes than hydroquinone.

Deoxyarbutin is a synthetic derivative of arbutin and is comparable to hydroquinone and arbutin in terms of TYR inhibitory activity. Deoxyarbutin is less cytotoxic to melanocytes than arbutin, as well as hydroquinone. [J. Dermatol. sci. vol 55(3), 179-184 (2009)]. Deoxyarbutin significantly improved fair human skin color when applied topically for 12 weeks. [Exp. Dermatol, vol 14(8), 601-608 (2005)].

Kojic acid is a chelating agent obtained during fungal fermentation in the production of soy sauce and sake, i.e. Japanese rice wine Kojic acid has been used to preserve or change color in food and cosmetic products. Kojic acid chelates to copper ions at the TYR active site to inhibit melanogenesis. The tautomerization of DOPAchrom to 5,6-dihydroxyindole-2-carboxylic acid is also inhibited. [J. Pharm. Pharmacol, vol 46(12), 982-985 (1994)]. Kojic acid has been widely used to treat melasma, although it can cause contact dermatitis, sensitization, and erythema. [J. Drugs Dermatol, vol 6(1), 32-39 (2007)].

Although there are a number of TYR inhibitors, their IC50 values compared to TYR are in most cases micromolar (µM). [J. Enzyme Inhibition Med. Chem. vol 32(1), 403-425 (2017)]. Such IC ₅₀ values are considered to be poor in view of their molecular size and increase the likelihood of cross-reactivity with other molecular target(s). Since a poor inhibitory activity is translated into a large transdermal dose, the inhibitory activity must be markedly improved in order to satisfy the desired transdermal activity against skin pigmentation as well as safety.

Pre-mRNA: Genetic information is carried on DNA (2-deoxyribonucleic 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 the exon and intron are interconnected with each other, as shown schematically below. Exons and introns are numbered as illustrated in the figure below.

Schematic description of the structure of pre-mRNA

Pre-mRNA splicing: Pre-mRNA is processed into mRNA after removal of introns by a series of complex reactions collectively referred to as "splicing", which is summarized schematically in the diagram below. [Ann. Rev. Biochem. 72(1), 291-336 (2003); Nature Rev. Mol. Cell biol. 6(5), 386-398 (2005); Nature Rev. Mol. Cell biol. 15(2), 108-121 (2014)].

Splicing is initiated by the formation of a "splicesome E complex" (ie, an early splice complex) between pre-mRNA and splicing adapter factors. In the "splicesome E complex", U1 binds to the junction of exon N and intron N, and U2AF ³⁵ binds to the junction of intron N and exon (N+1). Thus, the exon/intron or intron/exon junction regions are critical for the formation of the early splicesome complex. The "splicesome E complex" is converted to the "splicesome A complex" by additional complexation with U2. The "splicesome A complex" undergoes a series of complex reactions to remove or splice an intron to add adjacent exons.

Ribosomal protein synthesis: Proteins are encoded by DNA (2-deoxyribonucleic acid). In response to cellular stimulation or spontaneously, DNA is transcribed to produce pre-mRNA (pre-matrix ribonucleic acid) in the nucleus. The pre-mRNA introns are enzymatically spliced to produce mRNA (messenger ribonucleic acid), which is then translocated into the cytoplasm. In the cytoplasm, the translational machinery complex caused by the ribosome binds to the mRNA and protein synthesis occurs as it scans the genetic information encoded by the mRNA. [Biochemistry vol 41, 4503-4510 (2002); Cancer Res. vol 48, 2659-2668 (1988)].

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

If ASO binds tightly to mRNA in the cytoplasm, for example, ASO can inhibit ribosomal protein synthesis from mRNA. It is necessary for ASO to be present within the cytoplasm in order to inhibit ribosomal protein synthesis of its target protein.

Antisense Splicing Inhibition: If ASO binds tightly to pre-mRNA in the nucleus, ASO will be able to inhibit or modulate pre-mRNA splicing into mRNA. It is necessary for ASO to be present within the nucleus in order to inhibit or modulate pre-mRNA splicing into mRNA. This antisense splicing inhibition produces mRNA or mRNAs that do not contain the ASO-targeted exon. Such mRNA(s) are referred to as "splice variant(s)", and encode(s) for protein(s) smaller(s) than the protein encoded by the full-length mRNA.

In principle, splicing can be disrupted by inhibiting the formation of the "splicesome E complex". If ASO binds strongly to the exon-intron junction (5'→3'), i.e. "5' splicing site", then ASO blocks the formation of a complex between the pre-mRNA and factor U1, and hence the formation of the "splicesome E complex". Similarly, the "splicesome E complex" cannot be formed if ASO binds tightly to the junction region (5'→3') intron-exon, i.e. "3' splicing site".

The 3' splicing site and the 5' splicing site are schematically illustrated in the figure below.

Non-natural Oligonucleotides: DNA or RNA oligonucleotides are susceptible to degradation by endogenous nucleases, limiting their therapeutic value. Currently, many types of non-naturally occurring (non-naturally occurring) oligonucleotides have been developed and intensively researched. [clin. Exp. Pharmacol. Physiol, vol 33, 533-540 (2006)]. Some of them show long-term metabolic stability compared to DNA and RNA. Below are the chemical structures for several typical non-naturally occurring oligonucleotides. Such oligonucleotides bind predictably to a complementary nucleic acid, as does DNA or RNA.

B: Nucleic base

Phosphorothioate oligonucleotide: Phosphorothioate oligonucleotide (PTO) (Phosphorothioate oligonucleotide, PTO) is a DNA analog with one backbone of phosphate oxygen atoms replaced by a sulfur atom in the monomer. Such a small structural change made PTO relatively resistant to degradation by nucleases. [Ann. Rev. Biochem. vol 54, 367-402 (1985)].

Reflecting the structural similarity in the backbone of PTO and DNA, both of them poorly penetrate the cell membrane in most mammalian cell types. However, for some cell types overexpressing the DNA transporter(s), DNA and PTO show good cell penetration. Systemically administered PTO(s) are known to be readily distributed in the liver and kidneys. [Nucleic Acids Res. vol 25, 3290-3296 (1997)].

In order to facilitate the penetration of PTO into cells in vitro, lipofection is usually used in practice. However, lipofection physically alters the cell membrane, causes cytotoxicity, and therefore would not be ideal for long-term in vivo therapeutic applications.

Over the past 30 years, antisense PTO(s) and variants of PTO(s) have been clinically evaluated for the treatment of cancer, immunological disorders, metabolic diseases, and so on. [Biochemistry vol 41, 4503-4510 (2002); Clin. Exp. Pharmacol. Physiol, vol 33, 533-540 (2006)]. Many of these candidate antisense agents have not been successfully developed partly because of poor cell penetration of PTOs. In order to overcome poor cell penetration, PTO must be administered at a high dose for therapeutic activity.

However, PTO(s) are known to be associated with dose-limiting toxicity including prolonged clotting time, complement activation, tubular nephropathy, Kupffer cell activation and immune stimulation including splenomegaly, lymphoid hyperplasia, mononuclear cell infiltration. [clin. Exp. Pharmacol. Physiol, vol 33, 533-540 (2006)].

Many antisense PTO(s) have been found to show adequate clinical activity in diseases with significant hepatic or renal involvement. Mipomersen is a PTO analog that inhibits the synthesis of apoB-100, a protein involved in the transport of low-density lipoprotein (LDL) cholesterol. Mipomersen showed adequate clinical activity in patients with atherosclerosis, most likely due to its selective distribution in the liver. [Circulation vol 118(7), 743-753 (2008)]. ISIS-113715 is an antisense analog of PTO that inhibits the synthesis of protein tyrosine phosphatase IB (PTP1B) and has been found to show therapeutic activity in patients with type II diabetes mellitus. [Curr. Opin. Mol. Ther. vol 6, 331-336 (2004)].

Locked nucleic acid: In a locked nucleic acid (LNA), the ribose ring of the RNA backbone makes it structurally difficult to increase the binding capacity for RNA or DNA. Thus, LNA can be considered as a high affinity analogue of DNA or RNA. [Biochemistry vol 45, 7347-7355 (2006)].

Phosphorodiamidate morpholino oligonucleotide: In the phosphorodiamidate morpholino oligonucleotide (PMO), the phosphate backbone and 2-deoxyribose DNA are replaced by phosphoramidate and morpholine, respectively. [Appl. microbiol. Biotechnol. vol 71, 575-586 (2006)]. In this case, the DNA backbone is negatively charged, while the FMO backbone is not charged. Thus, the binding between FMO and mRNA has no electrostatic repulsion between backbones and is generally stronger than that between DNA and mRNA. Since PMO is structurally very different from DNA, PMO will not be recognized by the hepatic transporter(s) that recognizes DNA or RNA. At the same time, FMO does not easily penetrate the cell membrane.

Peptide Nucleic Acid: Peptide nucleic acid (PNA) is a polypeptide with N-(2-aminoethyl)glycine as a backbone and was discovered by Dr. Nielsen and colleagues. [Science vol 254, 1497-1500 (1991)]. The chemical structure and abbreviated nomenclature of PNA are illustrated in the figure below. Like DNA and RNA, PNA also selectively binds to a complementary nucleic acid. [Nature (London) vol 365, 566-568 (1992)]. When bound to a complementary nucleic acid, the N-terminus of the PNA is considered equivalent to the "5' end" of DNA or RNA and the C-terminus of PNA is considered to be the equivalent of the "3' end" of DNA or RNA.

Like FMO, the PNA backbone is not charged. Thus, the binding between PNA and RNA is generally stronger than the binding between DNA and RNA. Since PNA is markedly different from DNA in chemical structure, PNA will not be recognized by the hepatic DNA-recognizing transporter(s) and will show a tissue distribution profile different from that of DNA or PTO. However, PNA also poorly penetrates the membrane of mammalian cells. (Adv. Drug Delivery Rev. vol 55, 267-280, 2003).

Modified Nucleic Bases to Improve PNA Membrane Permeability: PNA has been made highly membrane permeable to mammalian cells by introducing modified nucleobases with a cationic lipid or equivalent covalently attached to it. The chemical structures of such modified nucleic bases are given below. These modified nucleobases of cytosine, adenine, and guanine have been found to hybridize predictably and complementarily to guanine, thymine, and cytosine, respectively. [PCT Appl. no. PCT/KR2009/001256; EP2268607; US8680253].

The incorporation of such modified nucleobases into PNA is similar to situations of lipofection. In lipofection, the phosphate backbone oligonucleotide molecules are obscured by cationic lipid molecules such as lipofectamine, and such lipofectamine/oligonucleotide complexes tend to penetrate the membrane rather easily compared to naked oligonucleotide molecules.

In addition to good membrane permeability, these PNA derivatives have been found to have an ultra-strong affinity for the complementary nucleic acid. For example, the introduction of 4 to 5 modified nucleic bases into 11-13-mer PNA derivatives easily gave rise to a T _m of 20° C. or more when duplexed with complementary DNA. Such PNA derivatives are very sensitive to base pair mismatch. The base pair mismatch resulted in a T _m loss of 11 to 22°C, depending on the type of modified base as well as the PNA sequence.

Small interfering RNA (siRNA) (small Interfering RNA, siRNA): Small interfering RNA (siRNA) refers to double-stranded RNA of 20-25 base pairs. [Microbiol. Mol. Biol. Rev. vol 67(4), 657-685 (2003)]. The antisense strand of siRNA somehow interacts with proteins, forming an “RNA-induced Silencing Complex” (RISC). The RISC then binds to a specific portion of the mRNA that is complementary to the siRNA antisense strand. mRNA complexed with RISC is cleaved. Thus, siRNA catalytically induces cleavage of its target (target) mRNA, and therefore suppresses protein expression by mRNA. RISC does not always bind to the full complementary sequence within the target mRNA, which raises concerns about non-targeted siRNA drug actions. Like other classes of DNA or RNA backbone oligonucleotide, siRNA has poor cell permeability and therefore tends to show poor in vitro or in vivo therapeutic activity unless properly prepared or chemically modified to obtain good membrane permeability.

Tyrosinase siRNA(s): TYR siRNA targeting the 19mer RNA sequence in human TYR mRNA was found to inhibit TYR mRNA and protein expression in human epidermal melanocytes (HEMs) after lipofection at 20 nM. siRNA suppressed TYR mRNA and protein, but did not affect the expression of tyrosinase-dependent proteins 1 and 2. siRNA inhibited UV-induced melanogenesis. [WWII Reports, vol 42(3), 178-183 (2009)].

siRNA(s) targeting mouse TYR mRNA were screened for their ability to suppress TYR mRNA and protein expression and melanogenesis in mouse melanoma B16 cells after lipofection at 40 nM. The most effective siRNA was injected into the eyeball of a C57BL/6 mouse and TYR mRNA inhibition in the retina was found to be approximately 60%. [Mol. Biol. International, vol 2010, Article ID 240472 (2010)].

Description of the invention

Problem being solved

There are other public examples for siPHK TYR. For example, the prior art discloses siRNA(s) directed to human TYR mRNA for the treatment of hyperpigmentation. [US 7504385]. However, the applicability of such siRNA(t) is questionable for use against hyperpigmentation. siRNA(s) are too expensive to manufacture and their cosmetic utility is generally limited. They must also be delivered to the skin via transdermal administration with proper user compliance. It should be anticipated that transdermal delivery is a huge technical challenge in the field of oligonucleotide.

Solution

This invention provides a peptide nucleic acid derivative represented by formula I, or a pharmaceutically acceptable salt thereof:

[Formula I]

where

n is an integer from 10 to 21;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

a compound of formula I is fully complementary to human pre-TYR mRNA or partially complementary to human pre-TYR mRNA with one or two mismatches from the total compound of formula I;

, S_n-1, S_n, Т₁, T₂,

, T_n-1 и T_n независимо обозначают атом дейтерия, атом водорода, замещенную или незамещенную алкилгруппу или замещенную или незамещенную арилгруппу; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n independently represent a deuterium atom, a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group;

X and Y independently represent a deuterium atom, a hydrogen atom [H], a formyl group [H-C(=O)-], an aminocarbonyl group [NH ₂ -C(=O)-], an aminothiocarbonyl group [NH ₂ -C(=S)- ], substituted or unsubstituted alkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted alkyl acyl group, substituted or unsubstituted arylacyl group, substituted or unsubstituted alkyloxycarbonyl group, substituted or unsubstituted aryloxycarbonyl group, substituted or unsubstituted alkylaminocarbonyl group, substituted or unsubstituted arylaminocarbonyl group, substituted or unsubstituted arylaminocarbonyl group, substituted or unsubstituted arylaminocarbonyl group, substituted or unsubstituted arylaminocarbonyl group, substituted or unsubstituted arylaminocarbonyl group, an arylaminothiocarbonyl group, a substituted or unsubstituted alkyloxythiocarbonyl group, a substituted or unsubstituted aryloxythiocarbonyl group, a substituted or unsubstituted alkylsulfonyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkylphosphonyl group, or a substituted or unsubstituted arylphosphonyl group;

Z is a hydrogen atom, a hydroxy group, a substituted or unsubstituted alkyloxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted amino group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group;

, B_n-1 и B_n независимо выбраны из природных нуклеиновых оснований, включая аденин, тимин, гуанин, цитозин и урацил, и нуклеиновых оснований не природного происхождения; и _B1 , _B2 ,

, B _n-1 and B _n are independently selected from naturally occurring nucleobases including adenine, thymine, guanine, cytosine, and uracil, and non-naturally occurring nucleobases; and

, B_n-1 и B_n независимо выбраны из нуклеиновых оснований не природного происхождения с замещенной или незамещенной аминогруппой, ковалентно связанной с остатком нуклеинового основания.at least four of B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from non-naturally occurring nucleic bases with a substituted or unsubstituted amino group covalently linked to the rest of the nucleic base.

The compound of formula I induces "exon 2" skipping in pre-mRNA human TYR, gives rise to splice variant(s) of human TYR mRNA that does not contain(s) "exon 2", and is therefore useful for inhibiting the functional activity of the pre-reading gene. human TYR mRNA.

The condition that "n is an integer from 10 to 21" literally means that n is an integer selected from the group of integers 11, 12, 13, 14.15, 16, 17.18, 19 and 20.

The chemical structures of naturally occurring or non-naturally occurring nucleobases in the PNA derivative of formula I are illustrated by the examples in FIG. 1a-1c. Natural (ie, naturally occurring) or non-naturally occurring (ie, not naturally occurring) nucleobases described herein include, but are not limited to, the nucleobases shown in FIG. 1a-1c. The provision of such non-naturally occurring nucleobases is illustrative of the variety of acceptable nucleobases and should therefore not be construed as limiting the scope of the present invention.

The substituents chosen to describe the PNA derivative of Formula I are shown in FIG. 2a-2e. Fig. 2a gives examples for substituted or unsubstituted alkyl groups. Substituted or unsubstituted alkyl acyl groups and substituted or unsubstituted arylacyl groups are shown in FIG. 2b. Fig. 2c illustrates examples for a substituted or unsubstituted alkylamino group, a substituted or unsubstituted arylamino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkylsulfonyl group, or an arylsulfonyl group. Fig. 2d gives examples of a substituted or unsubstituted alkyloxycarbonyl group or an aryloxycarbonyl group, a substituted or unsubstituted alkylaminocarbonyl group, or an arylaminocarbonyl group. In FIG. 2e are examples of a substituted or unsubstituted alkylaminothiocarbonyl group, a substituted or unsubstituted arylaminothiocarbonyl group, a substituted or unsubstituted alkyloxythiocarbonyl group, and a substituted or unsubstituted aryloxythiocarbonyl group. The provision of such exemplary substituents is illustrative of the variety of acceptable substituents and should therefore not be construed as limiting the scope of the present invention. A person skilled in the art can easily guess that the oligonucleotide sequence is a key factor for the sequence-specific binding of the oligonucleotide to the target pre-mRNA sequence, compared to substituents at the N-terminus or C-terminus.

The compound of formula 1 binds strongly to complementary DNA, an example of which is given in the prior art [PCT/KR2009/001256]. The duplex between the PNA derivative of Formula I and its full-length complementary DNA or RNA has a T _m value too high to reliably detect in aqueous buffer. The PNA compound of formula I gives high T _m values with shorter length complementary DNAs.

AUUGUCU], где последовательность интрона и экзона определяется соответственно "строчными" и "заглавными" буквами и область соединения интрона-экзона выражается "

". Традиционное обозначение для пре-мРНК дополнительно иллюстрируется 30-мерной последовательностью [(5'→3') ggguguuuuguacag

AUUGUCUGUAGCCGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека. Кроме того, 30-мерная последовательность пре-мРНК однозначно определяет 3' сайт сплайсинга, направленный соединением формулы I в пре-мРНК TYR человека, хотя нумерация экзонов TYR может описываться иным образом.The compound of formula I binds complementarily to the 3' splicing site of "exon 2" of human pre-mRNA TYR. [NCBI Reference Sequence: NG_0008748]. The 14-mer sequence [(5'→3') UGUACAGAUUGUCU] spans the junction of "intron 1" and "exon 2" in human pre-mRNA TYR, and consists of a 7-mer from "intron 1" and a 7-mer from " exon 2". Thus, a 14-mer pre-mRNA sequence can be commonly referred to as [(5'→3') uguacag

AUUGUCU], where the intron and exon sequence is defined by "lowercase" and "capital" letters, respectively, and the intron-exon junction is expressed as "

". The traditional notation for pre-mRNA is further illustrated by the 30-mer sequence [(5'→3') ggguguuuuguacag

AUUGUCUGUAGCCGA], encompassing the "intron 1" and "exon 2" junction in human pre-mRNA TYR. In addition, the 30mer pre-mRNA sequence uniquely defines the 3' splicing site targeted by the compound of formula I in human TYR pre-mRNA, although TYR exon numbering may be described differently.

The compound of formula I binds tightly to the target 3' splicing site of the human TYR pre-mRNA read from the human TYR gene and prevents the formation of an "early splicesome complex" including the compound's target exon. Since the compound of the present invention sterically inhibits the formation of the "early splice complex", TYR "exon 2" is spliced to produce splice variant(s) of TYR mRNA that does not contain "exon 2". Therefore, the compound of the present invention induces TYR "exon 2" skipping.

The strong affinity of the RNA allows the compound of formula I to cause TYR "exon 2" skipping even if the PNA derivative has one or two mismatches with the target 3' splicing site in the TYR pre-mRNA. Similarly, a PNA derivative of Formula I can still induce TYR "exon 2" skipping in a mutant TYR pre-mRNA having one or two single nucleotide polymorphisms (SNPs) at the target splicing site.

The compound of formula I has good cell permeability and can be easily delivered into the cell as a "naked" oligonucleotide, an example of which is the prior art [PCT/KR2009/001256]. Thus, the compound of the present invention induces "exon 2" skipping in TYR pre-mRNA and produces TYR mRNA splice variant(s) lacking TYR "exon 2" in cells treated with a compound of formula I as naked oligonucleotide. The compound of formula I does not require any means or formulations to be delivered to the cell to potently induce target exon skipping in cells. The compound of formula I readily induces TYR "exon 2" skipping in cells treated with a compound of the present invention as a "naked" oligonucleotide at a subfemtomolar concentration.

Due to good cellular or membrane permeability, the PNA derivative of formula I can be topically administered as a "naked" oligonucleotide to induce TYR "exon 2" skipping in the skin. The compound of formula I does not require a dosage form to increase transdermal delivery to the target tissue in terms of proper therapeutic or biological activity. Typically, a compound of formula I is dissolved in water and a co-solvent and topically or transdermally administered at a sub-picomolar concentration to elicit the desired therapeutic or biological activity in the target skin. The compound described in the present invention need not be administered vigorously or aggressively to elicit topical therapeutic activity. However, the PNA derivative of formula I may be formulated with cosmetic ingredients or adjuvants as a topical cream or lotion. Such a topical cosmetic cream or lotion may be useful in the treatment of facial hyperpigmentation.

The compound of Formula I may be used in combination 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 others.

The PNA derivative of formula I, or a pharmaceutically acceptable salt thereof, may be administered to the subject along with a pharmaceutically acceptable adjuvant, including, but not limited to, citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethylene glycol, polypropylene glycol, ethanol, isopropanol, sodium bicarbonate, distilled water, preservative(s) and others.

The compound described in the present invention may be administered topically to a subject at a therapeutically or biologically effective concentration ranging from 1 aM to more than 1 nM, which will vary depending on the dosing regimen, conditions or situations of the subject, and so on.

Preferred is a PNA derivative of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 10 to 21;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

a compound of formula I is fully complementary to human pre-TYR mRNA or partially complementary to human pre-TYR mRNA with one or two mismatches from the total compound of formula I;

, S_n-1, S_n, T₁, T₂,

, T_n-1 и T_n независимо обозначают атом дейтерия или атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n independently represent a deuterium atom or a hydrogen atom;

X and Y are independently deuterium atom, hydrogen atom, formyl group, aminocarbonyl group, aminothiocarbonyl group, substituted or unsubstituted alkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted alkylacyl group, substituted or unsubstituted arylacyl group, substituted or unsubstituted alkyloxycarbonyl group, substituted or unsubstituted aryloxycarbonyl group, substituted алкиламинокарбонилгруппу, замещенную или незамещенную ариламинокарбонилгруппу, замещенную или незамещенную алкиламинотиокарбонилгруппу, замещенную или незамещенную ариламинотиокарбонилгруппу, замещенную или незамещенную алкилокситиокарбонилгруппу, замещенную или незамещенную арилокситиокарбонилгруппу, замещенную или незамещенную алкилсульфонилгруппу, замещенную или незамещенную арилсульфонилгруппу, замещенную или незамещенную алкилфосфонилгруппу или замещенную или незамещенную арилфосфонилгруппу;

Z is a hydrogen atom, a hydroxy group, a substituted or unsubstituted alkyloxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted amino group;

, B_n-1, B_n независимо выбраны из природных нуклеиновых оснований, включая аденин, тимин, гуанин, цитозин и урацил, и нуклеиновых оснований не природного происхождения; _B1 , _B2 ,

, B _n-1 , B _n are independently selected from naturally occurring nucleobases including adenine, thymine, guanine, cytosine, and uracil, and non-naturally occurring nucleobases;

, B_n-1, B_n, независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV:at least four of B ₁ , B ₂ ,

, B _n-1 , B _n , are independently selected from non-naturally occurring nucleic bases represented by formula II, formula III or formula IV:

where

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from a hydrogen atom and a substituted or unsubstituted alkyl group;

L ₁ , L ₂ and L ₃ are a covalent linker represented by formula V, covalently connecting the basic amino group with the remainder of the nucleic base:

where

Q ₁ and Q _m represent a substituted or unsubstituted methylene group (-CH ₂ -) and Q _m directly associated with the basic amino group;

, Q_m-1, независимо выбраны из замещенной или незамещенной метиленгруппы, атома кислорода (-O-), атома серы (-S-) и замещенной или незамещенной аминогруппы [-N(H)- или -N(заместитель)-]; и _Q2 , _Q3 ,

, Q _m-1 independently selected from a substituted or unsubstituted methylene group, an oxygen atom (-O-), a sulfur atom (-S-) and a substituted or unsubstituted amino group [-N(H)- or -N(substituent)-]; and

m is an integer from 1 to 15.

Of interest is the PNA oligomer of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 11 to 18;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

the compound of formula I is fully complementary to human pre-mRNA TYR;

, S_n-1, S_n, T₁, T₂,

, T_n-1 и T_n представляют собой атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n represent a hydrogen atom;

X and Y independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl acyl group, a substituted or unsubstituted arylacyl group, a substituted or unsubstituted alkyloxycarbonyl group, or a substituted or unsubstituted aryloxycarbonyl group;

Z is a substituted or unsubstituted amino group;

, B_n-1, B_n независимо выбраны из природных нуклеиновых оснований, включая аденин, тимин, гуанин, цитозин и урацил, и нуклеиновых оснований не природного происхождения; _B1 , _B2 ,

, B _n-1 , B _n are independently selected from naturally occurring nucleobases including adenine, thymine, guanine, cytosine, and uracil, and non-naturally occurring nucleobases;

, B_n-1, B_n независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV;at least four of B ₁ , B ₂ ,

, B _n-1 , B _n are independently selected from non-naturally occurring nucleic bases represented by formula II, formula III, or formula IV;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from a hydrogen atom and a substituted or unsubstituted alkyl group;

Q ₁ and Q _m represent a substituted or unsubstituted methylene group and Q _m directly associated with the basic amino group;

и Q_m-1 независимо выбраны из замещенной или незамещенной метиленгруппы, атома кислорода и аминогруппы; и _Q2 , _Q3 ,

and Q _m-1 are independently selected from a substituted or unsubstituted methylene group, an oxygen atom, and an amino group; and

m is an integer from 1 to 11.

Of interest is a PNA derivative of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

the compound of formula I is fully complementary to human pre-mRNA TYR;

, S_n-1, S_n, T₁, T₂,

, , T_n-1 и T_n представляют собой атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, , T _n-1 and T _n represent a hydrogen atom;

X and Y are independently selected from a hydrogen atom, a substituted or unsubstituted alkylacyl group, or a substituted or unsubstituted alkyloxycarbonyl group;

Z is a substituted or unsubstituted amino group;

, B_n-1, B_n, независимо выбраны из природных нуклеиновых оснований, включая аденин, тимин, гуанин, цитозин и урацил, и нуклеиновых оснований не природного происхождения; _B1 , _B2 ,

, B _n-1 , B _n , are independently selected from naturally occurring nucleobases including adenine, thymine, guanine, cytosine, and uracil, and non-naturally occurring nucleobases;

, B_n-1, B_n, независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV;at least four of B ₁ , B ₂ ,

, B _n-1 , B _n , independently selected from non-naturally occurring nucleic bases represented by formula II, formula III or formula IV;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from a hydrogen atom and a substituted or unsubstituted alkyl group;

Q ₁ and Q _m is a methylene group and Q _m is directly linked to a basic amino group;

и Q_m-1 независимо выбраны из метиленгруппы, атома кислорода и аминогруппы; и _Q2 , _Q3 ,

and Q _m-1 are independently selected from a methylene group, an oxygen atom and an amino group; and

m is an integer from 1 to 9.

Of interest is the PNA oligomer of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

the compound of formula I is fully complementary to human pre-mRNA TYR;

, S_n-1, S_n, T₁, T₂,

, T_n-1 и T_n представляют собой атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n represent a hydrogen atom;

X and Y are independently selected from a hydrogen atom, a substituted or unsubstituted alkylacyl group, or a substituted or unsubstituted alkyloxycarbonyl group;

Z is a substituted or unsubstituted amino group;

, B_n-1 и B_n независимо выбраны из природных нуклеиновых оснований, включая аденин, тимин, гуанин, цитозин и урацил, и нуклеиновых оснований не природного происхождения;B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from naturally occurring nucleobases including adenine, thymine, guanine, cytosine, and uracil, and non-naturally occurring nucleobases;

, B_n-1 и B_n независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV;at least four of B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from non-naturally occurring nucleic bases represented by formula II, formula III or formula IV;

R ₁ , R ₃ and R ₅ represent a hydrogen atom and R ₂ , R ₄ and R ₆ independently represent a hydrogen atom or a substituted or unsubstituted alkyl group;

Q ₁ and Q _m are a methylene group and Q _m is directly linked to a basic amino group;

и Q_m-1 независимо выбраны из метиленгруппы, атома кислорода; и _Q2 , _Q3 ,

and Q _m-1 are independently selected from a methylene group, an oxygen atom; and

m is an integer from 1 to 9.

Of interest is a PNA derivative of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

the compound of formula I is fully complementary to human pre-mRNA TYR;

, S_n-1, S_n, T₁, T₂,

, T_n-1 и T_n представляют собой атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n represent a hydrogen atom;

X and Y are independently selected from a hydrogen atom, a substituted or unsubstituted alkylacyl group, or a substituted or unsubstituted alkyloxycarbonyl group;

Z is a substituted or unsubstituted amino group;

, B_n-1 и B_n независимо выбраны из аденина, тимина, гуанина, цитозина и нуклеиновых оснований не природного происхождения;B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from adenine, thymine, guanine, cytosine and non-naturally occurring nucleic bases;

, B_n-1 и B_n независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV;at least five of B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from non-naturally occurring nucleic bases represented by formula II, formula III or formula IV;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms;

Q ₁ and Q _m are a methylene group and Q _m is directly linked to a basic amino group;

и Q_m-1 независимо выбраны из метиленгруппы, атома кислорода; и _Q2 , _Q3 ,

and Q _m-1 are independently selected from a methylene group, an oxygen atom; and

m is an integer from 1 to 9.

Of interest is a PNA derivative of formula I or a pharmaceutically acceptable salt thereof:

where

n is an integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human TYR pre-mRNA;

the compound of formula I is fully complementary to human pre-mRNA TYR;

, S_n-1, S_n, T₁, T₂,

, T_n-1 и T_n представляют собой атом водорода; _S1 , _S2 ,

, S _n-1 , S _n , T ₁ , T ₂ ,

, T _n-1 and T _n represent a hydrogen atom;

X is a hydrogen atom;

Y is a substituted or unsubstituted alkylacyl group or a substituted or unsubstituted alkyloxycarbonyl group;

Z is a substituted or unsubstituted amino group;

, B_n-1 и B_n независимо выбраны из аденина, тимина, гуанина, цитозина и нуклеиновых оснований не природного происхождения; _B1 , _B2 ,

, B _n-1 and B _n are independently selected from adenine, thymine, guanine, cytosine and non-naturally occurring nucleic bases;

, B_n-1 и B_n независимо выбраны из нуклеиновых оснований не природного происхождения, представленных формулой II, формулой III или формулой IV;at least five of B ₁ , B ₂ ,

, B _n-1 and B _n are independently selected from non-naturally occurring nucleic bases represented by formula II, formula III or formula IV;

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms;

L ₁ denotes -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ group, -CH ₂ -O-(CH ₂ ) ₂ group, -CH ₂ -O-(CH ₂ ) ₃ group, -CH ₂ -O-(CH ₂ ) ₄ -group or -CH ₂ -O-(CH ₂ ) ₅ -group with the right end directly connected to the main amino group; and

L ₂ and L ₃ are independently selected from -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ group, -(CH ₂ ) ₃ -O-(CH ₂ ) ₂ group, -(CH ₂ ) ₂ -O -(CH ₂ ) ₃ groups, -(CH ₂ ) ₂ groups, -(CH ₂ ) ₃ groups, -(CH ₂ ) ₄ groups, -(CH ₂ ) ₅ groups, -(CH ₂ ) ₆ groups, -(CH ₂ ) ₇ groups and -(CH ₂ ) ₈ groups with the right end directly attached to the basic amino group.

Of interest is a PNA derivative of formula I, which is selected from the group of compounds below, or a pharmaceutically acceptable salt thereof:

(N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fethoc-AC(1/2)A(5)-GA(5)C-AA(5)T-CTG(6)-C(1/2)C-NH ₂ ;

(N→C) Ac-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Benzoyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Piv-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Methyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) n-Propyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fmoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) p-Toluenesulfonyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) H-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fethoc-Lys-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-Lys-NH ₂ ;

(N→C) Fethoc-Val-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Ac-Arg-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Benzyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Phenyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) [N-(2-Phenylethyl)amino]carbonyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Benzoyl-Leu-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Piv-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-Val-NH ₂ ;

(N→C) Fethoc -CA(7)G-AC(2O2)AA(4)TC(1O2)-TG(6)TA-NH ₂ ;

(N→C) Fethoc -CTG(6)-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fethoc -CA(5)G-ATA(5)-ATC(1O2)-TG(5)TA(5)-NH ₂ ;

(N→C) Fethoc -TA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ;

(N→C) Fethoc -TCA(3)-GAC(1O5)-A(5)AT-C(1O2)TG(5)-TA(5)-NH ₂ ;

(N→C) Fethoc -CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)C-NH ₂ ;

(N→C) Fethoc -AG(6)A-CA(5)A-TC(1O2)TG(6)TA(5)-C-NH ₂ ;

(N→C) Fethoc-AG(6)A-CA(5)A-TC(lO2)TG(6)TA(5)-CA(2O2)A-NH ₂ ;

(N→C) Fethoc -AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH ₂ ;

(N→C) Fethoc -AC(1O3)T-GA(6)C-TA(6)T-CTG(6)-TAC(1O5)-A(4)A-NH ₂ ;

(N→C) Fethoc-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH ₂ ;

(N→C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)CA(5)AA(2O2)-A-NH ₂ ;

(N→C) Fethoc-AA(6)T-CTG(6)-TA(6)CA(5)AA(2O2)-A-NH ₂ ;

(N→C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)CA(5)AA(2O3)-A-NH ₂ ;

(N→C) Fethoc-GC(1O4)T-AC(1O2)A-GA(4)C-AAT-CTG(6)-TA(6)C-NH ₂ ;

(N→C) Fethoc-GC(1O2)TA(3)CA-GA(4)C-AAT-C(1O2)TG-NH ₂ ;

(N→C) Fethoc-GC(1O2)TA(3)CA-G(2O2)AC-A(5)AT-C(1O2)TG-NH ₂ ; and

(N→C)Fethoc-GC(1O2)TA(3)CA-GA(4)CA(2O2)AT-C(1O2)TG-NH ₂ :

where

A, G, T and C are PNA monomers with natural nucleobase adenine, guanine, thymine and cytosine, respectively;

C(pOq), A(p), A(pOq), G(p) and G(pOq) are non-naturally occurring nucleobase PNA monomers represented by formula VI, formula VII, formula VIII, formula IX and formula X , respectively;

where

p and q are integers; and

abbreviations of N- and C-terminal substituents are specifically described as follows: "Fmoc-" is an abbreviation for "[(9-fluorenyl)methyloxy]carbonyl group";"Fethoc-" for the "[2-(9-fluorenyl)ethyl-1-hydroxy]carbonyl group";"Ac-" for "acetyl group";"Benzoyl-" for "benzenecarbonyl group";"Piv-" for "pivalyl group";"Methyl-" for "methyl group";"n-Propyl-" for "1-(n-propyl) group";"H-" for "hydrogen atom";"p-Toluenesulfonyl" for "(4-methylbenzene)-1-sulfonyl group";"-Lys-" for the amino acid residue "lysine";"-Val-" for the amino acid residue "valine";"-Leu-" for the amino acid residue "leucine";"-Arg-" for the amino acid residue "arginine";"-Gly-" for the amino acid residue "glycine";"[N-(2-Phenylethyl)amino]carbonyl-" for the "[N-1-(2-phenylethyl)amino]carbonyl group";"Benzyl-" for "1-(phenyl)methyl group";"Phenyl-" for "phenyl group";"Me-" for "methyl group"; and "-NH ₂ " for an unsubstituted "amino group".

Fig. 3 collectively and unambiguously represents the chemical structures for 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 [PCT/KR2009/001256], C(pOq) is regarded as a PNA monomer with "modified cytosine" due to its hybridization to "guanine". A(p) and A(pOq) are taken as PNA monomers with "modified adenine" when hybridized to "thymine". In the same way, G(p) and G(pOq) are considered as PNA monomers with "modified guanine" by base-pairing them with "cytosine".

Fig. 4 unambiguously illustrates the chemical structures for the various substituent abbreviations used to diversify the N-terminus or C-terminus of the PNA derivative of formula I in this invention.

To illustrate the abbreviations used for such PNA derivatives, the chemical structure for the 13-mer PNA derivative, abbreviated as "(N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG( 6)TA(5)-NH ₂ ", is shown in Fig.5 a. As another illustration, the chemical structure for the 15-mer PNA derivative, abbreviated "(N→C)

Fethoc-AG(6)A-CA(5)A-TC(1O2)TG(3O2)TA(5)-CA(2O2)A-NH ₂ " is shown in Fig. 5b.

AUUGUCUGUAGCCGA1. охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека.The 13mer PNA sequence "(N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ " shown in FIG. 5a is equivalent to the DNA sequence "(5'→3') CAG-ACA-ATC-TGT-A" for complementary binding to pre-mRNA. The 13mer PNA has a 13mer complementary overlap with the 13mer sequence indicated "bold" and "underlined" in the 30mer RNA sequence [(5'→3') ggguguuuuguacag

AUUGUCUGUAGCCGA1. spanning the junction region of "intron 1" and "exon 2" in human pre-mRNA TYR.

AUUGUCUGUAGCCGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека.The 15-mer PNA sequence "(N→C) Fethoc-AG(6)A-CA(5)A-TC(1O2)TG(3O2)TA(5)-CA(2O2)A-NH ₂ ", indicated on fig. 5b is equivalent to the DNA sequence "(5'→3') AGA-CAA-TCT-GTA-CAA" for complementary pre-mRNA binding. The 15mer PNA has a 15mer complementary overlap with the 15mer sequence indicated "bold" and "underlined" in the 30mer RNA sequence [(5'→3') ggguguuuuguacag

AUUGUCUGUAGCCGA], encompassing the "intron 1" and "exon 2" junction in human pre-mRNA TYR.

AU"U"GUC"U"GUAGCCGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека. Два ошибочных спаривания отмечены символом, заключенным в кавычки, т.е."".17-mer PNA sequence "(N→С) Fethoc-AC(1O3)T-GA(6)C-TA(6)T-CTG(6)-TAC(1O5)-A(4)A-NH ₂ " equivalent to the DNA sequence "(5'→3') ACT-GAC-TAT-CTG-TAC-AA" for complementary binding to pre-mRNA. The 17-mer PNA has a 15-mer complementary overlap with the 15-mer sequence indicated "bold" and "underlined" in the 30-mer RNA sequence [(5'→3') ggguguuuuguacag

AU"U"GUC"U"GUAGCCGA], encompassing the "intron 1" and "exon 2" junction in human pre-mRNA TYR. Two mismatches are marked with a character enclosed in quotation marks, i.e. "".

One aspect of the present invention is a pharmaceutical composition for the treatment of diseases or conditions associated with the expression of a human tyrosinase gene, comprising a peptide nucleic acid derivative in accordance with the present invention.

One aspect of the present invention is a pharmaceutical composition for the treatment of hyperpigmentation comprising a peptide nucleic acid derivative according to the present invention. Examples of hyperpigmentation caused by melanin oversynthesis include melasma, acne scars, liver spots, lentigo, age spots, and actinic damage.

One aspect of the present invention is a method of treating diseases or conditions associated with human tyrosinase gene expression, comprising administering a peptide nucleic acid derivative according to the present invention. The route of administration may be, for example, topical application.

One aspect of the present invention is a method for treating hyperpigmentation comprising administering a peptide nucleic acid derivative according to the present invention. The route of administration may be, for example, topical application.

One aspect of the present invention is the use of a peptide nucleic acid derivative according to the present invention in the preparation of a medicament for the treatment of diseases or conditions associated with the expression of a human tyrosinase gene.

One aspect of the present invention is the use of a peptide nucleic acid derivative according to the present invention in the preparation of a medicament for the treatment of hyperpigmentation. Brief decoding of figures

Fig. 1a-1c. Examples of naturally occurring or non-naturally occurring (modified) nucleic bases selected for the peptide nucleic acid derivative of Formula I.

Fig. 2a-2e. Examples of substituents selected for the peptide nucleic acid derivative of Formula I.

Fig. 3. Chemical structures of PNA monomers with a natural or modified nucleic base.

Fig. 4. Chemical structures for abbreviations of N- or C-terminal substituents.

Fig. 5a. Chemical structure of the 13-mer PNA derivative "(N→С) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ".

Fig. 5b. Chemical structures of the 15-mer PNA derivative "(N→С) Fethoc-AG(6)A-CA(5)A-TC(1O2)TG(3O2)TA(5)-CA(2O2)A-NH ₂ ".

Fig. 6. Chemical structures of the Fmoc-PNA monomers used to synthesize the PNA derivatives of the present invention.

Fig. 7a-7b. C ₁₈ reverse phase HPLC chromatograms of "ASO 1" before and after HPLC purification, respectively.

Fig. 8. ESI-TOF mass spectrum of "ASO 1" purified C ₁₈ -RP by preparative HPLC.

Fig. 9a. Electrophoretic analysis of nested PCR products in B16F10 mouse melanoma cells treated with 0 (negative control), 1.10 or 1000 aM "ASO 1M".

Fig. 9b. Sanger sequencing data for the PCR product attributed to exon skipping (2-3).

Fig. 10a. Changes in full-length TYR mRNA level by qPCR in B16F10 mouse melanoma cells treated with "ASO 1M" at 0 (negative control), 1, 10, 100, or 1000 aM (standard error).

Fig. 10b. Western blot data on TYR in B16F10 mouse melanoma cells treated for 24 hours with "ASO 1M" at 0 zM (negative control), 10 zM, 100 zM, 1 aM, or 10 aM (i.e., 10,000 zM).

Fig. 11. Changes in melanin content in B16F10 mouse melanoma cells treated with either "ASO 1M" at 1, 10, 100, or 1000 aM, or 10 μg/ml, or 100 μg/ml arbutin (error standard error).

Fig. 12. Changes in full length TYR mRNA level by qPCR in human primary epithelial melanocytes treated with "ASO 1" at 0 zM (negative control), 1 zM, 100 zM or 10 aM. (error value according to the standard error).

The best embodiment of the invention

General procedures for the preparation of PNA oligomers

PNA oligomers were synthesized using solid phase peptide synthesis (SPPS) based on Fmoc chemistry in accordance with the method disclosed in the prior art [US6,133,444; WO96/40685] with minor but required modifications. The solid support used in this study was H-Rink Amide-ChemMatrix amide resin purchased from PCAS BioMatrix Inc. (Quebec, Canada). Nucleic base-modified Fmoc-PNA monomers were synthesized as described in the prior art [PCT/KR 2009/001256] or with minor modifications. For the synthesis of the PNA derivatives of the present invention, nucleobase-modified Fmoc-PNA monomers and naturally occurring nucleobase Fmoc-PNA monomers have been used. PNA oligomers were purified by C^-reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

Scheme 2 illustrates a typical monomer elongation cycle adopted in the SPPS of this study, and the synthetic details (subtleties) are given below. However, for one skilled in the art, there are many minor variations obviously possible when such SPPS reactions are run efficiently on an automatic peptide synthesizer or a manual peptide synthesizer. Each reaction step in Scheme 2 is summarized as follows.

Scheme 2

[Activation of H-Rink-ChemMatrix Resin] 0.01 mmol (about 20 mg resin) of ChemMatrix resin in 1.5 ml of 20% piperidine/DMF was shaken in a "libra" tube for 20 minutes and the DeFmoc solution was filtered. The resin was washed for 30 seconds successively each time with 1.5 ml of methylene chloride (methylene chloride, MS), 1.5 ml of dimethylformamide (dimethylformamide, DMF), 1.5 ml of MS, 1.5 ml of DMF and 1.5 ml of MS . The resulting free amines on a solid support were reacted with either the Fmoc-PNA monomer or an Fmoc-protected amino acid derivative.

[DeFmoc] The resin was shaken in 1.5 ml of 20% piperidine/DMF for 7 minutes and the DeFmoc solution was filtered. The resin was washed for 30 seconds successively each time with 1.5 ml MS, 1.5 ml DMF, 1.5 ml MS, 1.5 ml DMF and 1.5 ml MS. The resulting free amines on a solid support were immediately reacted with the Fmoc-PNA monomer.

[Reaction with Fmoc-PNA Monomer] Free amines on a solid support were reacted with Fmoc-PNA monomer as follows. 0.04 mmol of PNA monomer, 0.05 mmol of HBTU and 10 mmol of DIEA were incubated for 2 min in 1 ml of anhydrous DMF and added to the resin with free amines. The resin solution was shaken for 1 hour and the reaction mixture was filtered. The resin was then washed for 30 seconds successively each time with 1.5 ml MS, 1.5 ml DMF and 1.5 ml MS. The chemical structures of the nucleobase-modified Fmoc-PNA monomers used in the present invention are shown in FIG. 6. The nucleobase-modified Fmoc-PNA monomers shown in FIG. 6 are to be taken as examples and therefore should not be construed as limiting the scope of the present invention. A person skilled in the art can easily guess a number of changes in the Fmoc-PNA monomers for the synthesis of the PNA derivative of formula I.

[Capping] After the coupling reaction, the unreacted free amines were capped with shaking for 5 minutes in 1.5 ml of a capped solution (5% acetic anhydride and 6% 2,6-leutidine in DMF). The capped solution was then filtered and washed for 30 sec each time successively with 1.5 ml MC, 1.5 ml DMF and 1.5 ml MC.

[Introduction of "Fethoc-" Group at N-Terminus] The "Fethoc-" group was introduced at the N-terminus by reacting the free amine on the resin with "Fethoc-OSu" under basic coupling conditions. Chemical structure of Fethoc-OSu [CAS No. 179337-69-0, C ₂₀ H ₁₇ NO ₅ , M.B. 351.36] is listed as below.

[Resin Removal] The resin-bound PNA oligomers were cleaved from the resin by shaking for 3 hours in 1.5 ml of a cleavage solution (2.5% tri-isopropylsilane and 2.5% 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 to purify by reverse phase HPLC.

[HPLC Analysis and Purification] After resin cleavage, the technical product of the PNA derivative was purified by C ₁₈ reverse phase HPLC eluting with water/acetonitrile or water/methanol (gradient method) containing 0.1% TFA. Fig. 7a and 7b are representative HPLC chromatograms for "ASO 1" before and after HPLC purification, respectively. The sequence of the "ASO 1" oligomer is as shown in Table 1.

Synthetic Examples for a PNA Derivative of Formula I

In order to complementarily target the 3' splicing site of "exon 2" in human TYR pre-mRNA, a PNA derivative of the present invention was prepared according to the synthetic methods above or with minor modifications. The provision of such PNA derivatives targeting human TYR pre-mRNA is an example of a PNA derivative of formula I and should not be construed as limiting the scope of the present invention.

Table 1 lists PNA derivatives that complementarily target the 3' splicing site of "exon 2" in human TYR pre-mRNA read from the human TYR gene [NCBI (National Center for Biotechnology Information) Standard/Reference Sequence: NG_0008748], as well as data on structural study using mass spectrometry. The provision of TYR ASOs in Table 1 is an example of PNA derivatives of formula I and should not be construed to limit the scope of the present invention.

AUUGU-CUGUAGCCGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека. Таким образом, "ASO 1" имеет 5-мерное перекрывание с "интроном 1" и 8-мерное перекрывание с "экзоном 2" в пределах пре-мРНК TYR человека."ASO 1" has a 13-mer complementary overlap with the 13-mer sequence indicated "bold" and "underlined" within the 30-mer RNA sequence [(5'→3') ggguguuuuguacag

AUUGU-CUGUAGCCGA], encompassing the "intron 1" and "exon 2" junction in human pre-mRNA TYR. Thus, "ASO 1" has a 5-mer overlap with "intron 1" and an 8-mer overlap with "exon 2" within human TYR pre-mRNA.

Table 2 lists "ASO 1M" complementarily targeting the "exon 2" 3' splicing site in mouse TYR pre-mRNA read from mouse genomic DNA [provided access to NCBI Standard/Reference Sequence: NC_000073], as well as structural data. research using mass spectrometry. Providing "ASO 1M" is to evaluate antisense activity in cells of murine origin.

AUCAUUUGUAGCAGA], охватывающую область соединения интрона 1 и экзона 2 в пре-мРНК TYR мыши, считывали с гена TYR мыши. [Номер доступа гена NCBI: NC_000073].30-mer RNA sequence [(5'→3') aauuguuuuucacag

AUCAUUUGUAGCAGA], spanning the junction of intron 1 and exon 2 in mouse TYR pre-mRNA, was read from the mouse TYR gene. [NCBI gene access number: NC_000073].

AUCAUUUGUAGCAGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR мыши."ASO 1M" is equivalent to the DNA sequence "(5'→3') CAA-ATG-ATC-TGT-G" for complementary binding to pre-mRNA. "ASO 1M" has a 13mer complementary overlap with the 13mer sequence indicated "bold" and "underlined" within the 30mer RNA sequence [(5'→3') aauuguuuuucacag

AUCAUUUGUAGCAGA], encompassing the "intron 1" and "exon 2" junction in mouse TYR pre-mRNA.

Like "ASO 1" against human pre-TYR mRNA, "ASO 1M" has a 5-mer overlap with "intron 1" and an 8-mer overlap with "exon 2" within mouse pre-TYR mRNA. "ASO 1M" can serve as a good substitute (mimic) compound for the antisense activity of "ASO 1" in human cells.

Fig. 7a is the HPLC chromatogram obtained for the technical (crude) product "ASO 1". The crude product was purified by C ₁₈ -RP preparative HPLC. Fig. 7b is the HPLC chromatogram for the purified "ASO 1" product. The purity of "ASO 1" improved markedly after purification by preparative HPLC. Fig. 8 shows the ESI-TOF mass spectrum obtained from the purified "ASO 1" product. The provision of analytical data for "ASO 1" is to illustrate how the PNA derivatives of formula I were purified and identified in the present invention and should not be construed as limiting the scope of the present invention.

Binding affinity of typical PNA derivatives for complementary DNA

The PNA derivatives in Tables 1 and 2 were evaluated by their binding affinity for 10-mer DNA(s) targeting either the N-terminus or the C-terminus complementarily. Binding affinity was assessed using the T _m value for the duplex between PNA and 10-mer complementary DNA. The duplex between PNA derivatives and fully complementary DNA(s) show T _m values too high to be reliably determined in an aqueous buffer solution, since the buffer solution tends to boil during T _m measurement.

The values of T _m were determined on a spectrometer with UV and visible regions of the spectrum as follows. A mixed solution of 4 µmol PNA oligomer and 4 µmol complementary 10-mer DNA in 4 ml aqueous buffer (pH 7, 16, 10 mmol sodium phosphate, 100 mmol NaCl) in a 15 ml Falcon polypropylene tube was incubated at 90°C for one minute and cooled slowly to normal temperature. The solution was then transferred to a 3 ml UV quartz cuvette fitted with a sealed lid and subjected to T _m measurement at 260 nm on a UV-Visible spectrophotometer as described in the prior art [PCT/KR2009/001256] or with minor modifications. 10mer complementary DNA(s) for T _m measurement were purchased from Bioneer (www.bioneer.com, Dajeon, Republic of Korea) and used without further purification.

The observed T _m values of PNA derivatives of formula I are very high for complementary binding to 10-mer DNA and are shown in Table 3. For example, "ASO 1" showed a T _m value of 78.0°C for a duplex with 10-mer complementary DNA , targeting the 10-mer N-terminus in PNA, designated "bold" and "underlined" in [(N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6 )TA(5)-NH ₂ ]. Simultaneously, "ASO 1" showed a T _m of 73.0°C for a duplex with 10-mer complementary DNA targeting the 10-mer C-terminus in PNA, indicated by "bold" and "underlined" in [( N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ].

Examples of biological activities of PNA derivatives of formula I

The PNA derivatives of the present invention were evaluated in vitro for TYR antisense activities in B16F10 mouse melanoma cells and human melanocytes. The biological examples are provided as examples to illustrate the biological profiles of the PNA derivatives of Formula I and should therefore not be construed as limiting the scope of the present invention.

Example 1 Exon skipping induced by "ASO 1M".

AUCAUUUGUAGCAGA], охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR мыши. Одновременно, "ASO 1М" обладает 9-мерной комплементарностью наряду с 4 ошибочными спариваниями, обозначенными "жирным шрифтом" и "подчеркнутых" в пределах 30-мерной последовательности РНК [(5'→3') ggguguuuug"u"acag

AU"UG"U"C"UGUAGCCGA1. охватывающей область соединения "интрона 1" и "экзона 2" в пре-мРНК TYR человека. 4 ошибочные спаривания отмечены кавычками (" ")."ASO 1M" precisely defined in Table 2 has a 13-mer complementary overlap with the 13-mer sequence indicated "bold" and "underlined" within the 30-mer RNA sequence [(5'→3') aauuguuuuucacag

AUCAUUUGUAGCAGA], encompassing the "intron 1" and "exon 2" junction in mouse TYR pre-mRNA. Simultaneously, "ASO 1M" has 9-mer complementarity along with 4 mismatches marked "bold" and "underlined" within the 30-mer RNA sequence [(5'→3') ggguguuuug"u"acag

AU"UG"U"C"UGUAGCCGA1. spanning the junction region of "intron 1" and "exon 2" in human pre-mRNA TYR. The 4 mismatches are marked with quotation marks ("").

"ASO 1M" was evaluated for its ability to induce TYR "exon 2" skipping in mouse B16F10 melanoma cells despite "ASO 1M" having 4 mismatches with the 3' "exon 2" splicing site in human TYR pre-mRNA. "ASO 1M", which is fully complementary to mouse pre-TYR mRNA, can serve as a good substitute (mimic) compound for "ASO 1", which is fully complementary to human pre-TYR mRNA. [Cell Culture and ASO Treatment] B16F10 mouse melanoma cells (Cat. Number CRL-6475, ATCC) were maintained in DMEM (Dulbecco's Modified Minimal Essential Eagle's Medium) supplemented with 10% FBS, 1% streptomycin/penicillin, and 0.01 mg /ml bovine insulin. B16F10 cells grown in a 60 mm culture dish containing 5 ml DMEM were incubated for 5 hours with "ASO 1M" at 0 (negative control), 1, 10, 100 or 1000 aM.

[RNA Extraction and cDNA Synthesis by One Step PCR] After incubation for 5 hours, total RNA was extracted with a "Universal RNA Extraction Kit" (Cat. Number 9767, Takara) according to the manufacturer's instructions. 200 ng messenger RNA was used for a 25 µl reverse transcription reaction using the Super ^Script® Platinum® ^Taq DNA Polymerase One-Step Reverse Transcription Polymerase Chain Reaction Kit (Cat. No. 10928-042, Invitrogen) against the exon-specific primer set [exon 1_straight: (5'→3') GTAAGTTTGGATTTGGGG; and exon 4_reverse: (5'→3') AGAGC-GGTATGAAAGGAA] according to the following cycle conditions: 50°C for 30 min and 94°C for 2 min followed by 15 cycles of 30 sec at 94°C, 30 sec at 52°C and 40 sec at 72°C.

[Nested PCR amplification] 1 µl cDNA was further amplified in 20 µl nested PCR (Cat. No. K2612, Bioneer) against the primer set [exon 1n_forward: (5'→3') GAGAACTAACTGGGGATGA; and exon 4n_reverse: (5'→3') CGATAGGTGCATTGGCTT] according to cycle conditions precisely defined as follows: 95°C for 5 min followed by 30 cycles of 30 sec at 95°C, 30 sec at 52°C and 40 sec at 72°C.

[Identification of exon skipping products] PCR products were subjected to electrophoretic separation on a 2% agarose gel. Bands of the required size were collected and analyzed by Sanger sequencing.

Fig. 9a shows the electrophoretic data of the PCR products. Cells without "ASO 1M" treatment gave two intense PCR bands, one for full-length TYR mRNA and the other for a splice variant of TYR mRNA lacking exons 2 and 3. Thus, exon skipping 2-3 is believed to occur spontaneously. Cells treated with "ASO 1M" at 1 to 1000 aM, however, produced only the splicing variant of TYR mRNA lacking exons 2 and 3. Thus, "ASO 1M" increased the propensity for skipping exons 2-3 in B16F10 melanoma cells. The exon skipping PCR product was sequenced to skip exons 2-3 as shown in FIG. 9b.

Example 2 qPCR for TYR mRNA in B16F10 mouse melanoma cells treated with "ASO 1M".

"ASO 1M" was evaluated by TYR nested qPCR for its ability to induce changes in mouse TYR mRNA levels in B16F10 cells, as described below.

[Cell Culture and ASO Treatment] B16F10 cells grown in a 60 mm culture dish containing 5 ml of DMEM were treated with "ASO 1M" at 0 (negative control), 1, 10,100 or 1000 aM (2 culture dishes per dose).

[RNA Extraction and cDNA Synthesis by One Step PCR] After incubation with "ASO 1M" for 5 hours, total RNA was extracted from the cells using the "Universal RNA Extraction Kit" (Cat. No. 9767, Takara) according to the manufacturer's instructions. 200 ng messenger RNA was used for a 25 µl reverse transcription reaction with the One-Step RT-PCR Kit (Invitrogen, USA) against the Exon Specific Primer Kit [exon 1 forward: (5'→3') GTAAGTTTGGATTTGGGG; and exon 4_reverse: (5'→3')

AGAGCGGTATGAAAGGAA] according to the following cycle conditions: 50°C for 30 min and 94°C for 2 min followed by 15 cycles of 30 sec at 94°C, 30 sec at 52°C and 40 sec at 72°C.

[Nested qPCR amplification] The cDNA solutions were diluted 100-fold and 1 µl of each diluted PCR product was reacted with 20 µl real-time PCR with a set of Taqman probes spanning the junction of exon 2 and exon 3 (Cat. No. Mm00495818_m1, Thermo Fisher Scientific) according to the following cycle conditions: 95°C for 3 minutes followed by 30 cycles of 10 seconds at 95°C and 30 seconds at 60°C.

[Statistical Analysis] The nested qPCR experiment was independently repeated 4 times and the individual mDNA levels from each experiment were normalized against the mDNA level without "ASO 1M" treatment. mDNA levels obtained from all 4 separate experiments were pooled for statistical analysis using a parametric Student's t-test. Thus, the number of RNA samples is 8 for each concentration.

Fig. 10a shows pooled qPCR data in which the level of full-length mDNA was significantly reduced by 40% in cells treated with "ASO 1M" at 1 to 1000 aM.

Example 3 Inhibition of TYR protein expression in B16F10 melanoma cells with "ASO 1M".

"ASO 1M" was evaluated for its ability to suppress TYR protein expression in B16F10 mouse melanoma cells, as described below.

B16F10 cells grown in a 60 mm culture dish containing 5 ml DMEM were treated with "ASO 1M" at 0 zM (negative control), 10 zM, 100 zM, 1 aM, or 10 aM. After 24 hours, cells were washed 2 times with cold phosphate buffered saline (PBS) and then lysed with 200 µl of 1X cell lysis buffer (Cat. No. 9803, Cell Signaling Tech) supplemented with a mixture of 1X protease inhibitors ( Cat. No. P8340, Sigma). 200 μl of each lysate in a 1.5 ml e-tube was mixed with 100 μl of 5X sample buffer and boiled at 100° C. for 5 minutes. 20 μl of the lysate was subjected to electrophoretic separation on a 4-15% gradient TGX gel (Cat No. 456-1086, Bio-Rad) and the protein was transferred to a 0.45 μm PVDF membrane. The membrane was tested with an anti-TYR antibody (Cat. No. 9319, Cell Signaling Tech) and an anti-β-actin antibody (Cat. No. a3845, Sigma).

Fig. 10b shows Western blot data for TYR protein expression in B16F10 cells. The intensity of the TYR expression band was significantly weaker in lysates treated with "ASO 1M" than in lysates without "ASO 1M" treatment; negative control. TYR protein and mRNA level decreased commensurately with ASO treatment. (cf. "Example 2").

Example 4 Inhibition of Melanogenesis in B16F10 Melanoma Cells with "ASO 1M"

"ASO 1M" was evaluated for its ability to inhibit melanogenesis in B16F10 mouse melanoma cells as described below.

B16F10 cells subcultured in a 60 mm culture dish containing 5 ml DMEM were treated untreated (negative control) and treated with "ASO 1M" at 1 to 1000 aM or with 10 or 100 µg/ml arbutin as a positive control (2 culture dishes per dose). After 24 hours, the cells were washed 2 times with cold phosphate-buffered saline and lysed with 200 μl of 1N NaOH. Each lysate was collected in a 1.5 ml e-tube and kept overnight at room temperature. The melanin content of each lysate was determined by absorbance at 475 nm on an ELISA reader. The same experiment was repeated four times using cells in different passages. Four sets of melanin content data were pooled for statistical analysis using a parametric Student's t-test against untreated melanin content (negative control).

Fig. 11 summarizes changes in melanin content in B16F10 cells after 24 hours of incubation with either "ASO 1M" or arbutin. Melanin content was significantly reduced by about 15% and 25% in cells treated with 10 µg/ml and 100 µg/ml arbutin, respectively. In the case of cells treated with "ASO 1M", the melanin content was significantly reduced by about 15% with no noticeable dose dependence. The inhibitory activity of "ASO 1M" was comparable to that of 10 μg/ml arbutin.

Example 5 Preparation of a topical cream containing a compound of formula I

A compound of formula I, eg "ASO 2", was formulated as a topical cream for subjects. Cream for topical use was prepared as described below. Given that there are a myriad of possible topical creams, this preparation is to be taken as an example and should not be construed as limiting the scope of the present invention.

[Preparation of Solution A] Distilled water (196 g), disodium EDTA (0.06 g), glycerin (15 g), dipropylene glycol (30 g), phenoxyethanol (0.9 g), ethylhexylglycerol (0.9 d) copolymer

sodium hydroxyethyl acrylate/acryloyldimethyl taurate (2.4 g) and 1 nM "ASO 2" aqueous solution (0.3 ml). The mixture is subjected to homogenization at 70 ~ 75°C using a homogenizer mixer.

[Preparation of Solution B] In another beaker, cetearyl ethylhexanoate (12g), dimethicone (6g), stearic acid/hydrogenated vegetable oil/behenyl alcohol/cetearyl alcohol (15g), shea butter (Shea ) (9 g) and polyglyceryl-3-methylglucose distearate/glyceryl stearate SE/methylglucose sesquistearate (6 g).

[Preparation of Topical Cream] "Solution A" and "Solution B" were mixed by subjecting to homogenization at 7200 rpm and 70 ~ 75°C for 3 minutes with a homogenizer mixer. The mixture was slowly cooled to room temperature to give a topical cream containing 1 pM "ASO 2".

Example 6 qPCR for TYR mRNA in human melanocytes treated with "ASO 1".

AUUGUCUGUAGCCGA]."ASO 1" in Table 1 represents the 13-mer ASO TYR fully complementary to the 3' splicing site spanning the intron 1-exon 2 junction in human TYR, indicated by "bold" and "underlined" in the 30-mer sequence of pre -human TYR mRNA [(5'→3') ggguguuuuguacag

AUUGUCUGUAGCCGA].

"ASO 1" was evaluated by TYR nested qPCR for its ability to induce changes in TYR mRNA levels in primary human epidermal melanocytes as follows.

[Cell Culture and ASO Treatment] Primary epidermal melanocyte cells (Cat. Number PCS-200-013, ATCC) were maintained in dermal cell basal medium (Cat Number PCS-200-030, ATCC) supplemented with part of the Mature Melanocyte Growth Kit ( Cat. Number PCS-200-042, ATCC). Melanocytes grown in a 60 mm culture dish containing 5 ml culture medium were treated with "ASO 1" at 0 zM (negative control), 1 zM, 100 zM, or 10 aM. (3 culture dishes for each concentration).

[RNA Extraction and cDNA Synthesis by One Step PCR] After incubation with "ASO 1M" for 5 hours, total RNA was extracted using "RNeasy Mini Kit" (Cat. Number 74106, Qiagen) according to the manufacturer's instructions. 200 ng messenger RNA was subjected to a 25 µl reverse transcription reaction using the Super Script ^® One-Step RT-PCR Kit with Platinum ^® Taq DNA Polymerase (Cat. No. 10928-042, Invitrogen) against the exon-specific primer set [exon 1_forward(2) : (5'→3') CTCTTTGTCTGGATGCATT; and exon 5_reverse: (5'→3') CTGTGGTAATCCTCTTTCT] according to the following specified cycle conditions: 50°C for 30 min and 94°C for 2 min followed by 15 cycles of 30 sec at 94°C, 30 sec at 50 °C and 1 min at 72°C.

[Nested PCR amplification] 1 µl cDNA was further amplified in 20 µl nested PCR reaction (Cat. No. K2612, Bioneer) against the exon-specific primer set [exon 2n_forward(2): (5'→3') GATAAAGCTGCCAATTTC; and exon 3n_reverse: (5'→3') TTGTGCATGCTGCTTTGA] versus the Taqman probe [(5'→3') 5,6-FAM-CACTGG-ZEN-AAGGATTTGCTAGTCCAC-3IABkFQ]. Cycle conditions: 95°C for 3 minutes followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C.

Fig. 12 shows qPCR data in which the level of full-length TYR mDNA decreased by about 30% in human melanocytes treated with "ASO 1" from 1 zM to 10 aM. The observed reductions were significant (parametric Student's t-test) in cells treated with "ASO 1" at 1 zM and 10 aM.

Claims (52)

1. A peptide nucleic acid derivative represented by formula I:

where n is an integer from 13 to 17; the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human tyrosinase pre-mRNA; a compound of formula I is fully complementary to human tyrosinase pre-mRNA or partially complementary to human tyrosinase pre-mRNA with one or two mismatches from the entire compound of formula I; S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n denote a hydrogen atom; X is a hydrogen atom [H]; Y is a substituted or unsubstituted alkyloxycarbonyl group wherein the substituted or unsubstituted alkyloxycarbonyl group is selected from:

Z is an amino group; _{B 1 ,} B ₂ . and at least four _{of B 1} , B ₂ , .

where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms; L ₁ , L ₂ and L ₃ are a covalent linker represented by formula V, covalently connecting the basic amino group with the remainder of the nucleic base:

where Q ₁ and Q _m represent a methylene group (-CH ₂ -) and Q _m directly binds the basic amino group; Q ₂ , Q ₃ , ..., and Q _m-1 represent a methylene group in which one of Q ₂ , Q ₃ , ... and Q _m-1 is independently selected from a methylene group and an oxygen atom (-O-); and m is an integer from 4 to 6. 2. A peptide nucleic acid derivative according to claim 1, where n is an integer from 13 to 15; the compound of formula I has at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence [(5'→3') UGUACAGAUUGUCU] in human tyrosinase pre-mRNA; the compound of formula I is fully complementary to human pre-mRNA tyrosinase; S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n represent a hydrogen atom; X is a hydrogen atom; Y is a substituted or unsubstituted alkyloxycarbonyl group wherein the substituted or unsubstituted alkyloxycarbonyl group is selected from:

Z is an amino group; B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from adenine, thymine, guanine, cytosine and non-naturally occurring nucleic bases; at least _five of _{B 1} , B ₂ , . R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen atoms; L ₁ denotes -(CH ₂ )2-O-(CH ₂ ) ₂ -, -CH ₂ -O-(CH ₂ ) ₂ -, -CH ₂ -O-(CH ₂ ) ₃ -, -CH ₂ -O -(CH ₂ ) ₄ - or -CH ₂ -O-(CH ₂ ) ₅ - with the right end directly associated with the basic amino group; and L ₂ and L ₃ are independently selected from -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -O-(CH ₂ ) ₂ -, -(CH ₂ ) ₂ -O-( CH ₂ ) ₃ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -, -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ - and -(CH ₂ ) ₈ - with the right end is directly associated with the basic amino group. 3. A peptide nucleic acid derivative according to claim 1, which is selected from the group of peptide nucleic acid derivatives below: (N→C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)TA(5)-NH ₂ ; and (N→C) Fethoc-AC(1O2)A(5)-GA(5)C-AA(5)T-CTG(6)-C(1O2)C-NH ₂ ; where A, G, T and C are PNA monomers with natural nucleobase adenine, guanine, thymine and cytosine, respectively; C(pOq), A(p), A(pOq), G(p) and G(pOq) are non-naturally occurring nucleobase PNA monomers represented by formula VI, formula VII, formula VIII, formula IX and formula X , respectively:

where p and q are integers from 1 to 8; and abbreviations of N- and C-terminal substituents are specifically described as follows: "Fethoc-" for "[2-(9-fluorenyl)ethyl-1-oxy]carbonyl group"; and "-NH ₂ " for an unsubstituted "amino group". 4. Pharmaceutical composition for the treatment of diseases or conditions associated with the expression of the human tyrosinase gene, comprising an effective amount of a peptide nucleic acid derivative according to claim 1. 5. Pharmaceutical composition for the treatment of hyperpigmentation associated with the expression of the human tyrosinase gene, comprising an effective amount of a peptide nucleic acid derivative according to claim 1. 6. A method of treating diseases or conditions associated with the expression of the human tyrosinase gene, which includes the introduction of a peptide nucleic acid derivative according to claim 1. 7. A method for treating hyperpigmentation associated with human tyrosinase gene expression, comprising the administration of a peptide nucleic acid derivative according to claim 1. 8. The use of a peptide nucleic acid derivative according to claim 1 in the preparation of a medicament for the treatment of diseases or conditions associated with the expression of the human tyrosinase gene. 9. The use of a peptide nucleic acid derivative according to claim 1 in the preparation of a medicament for the treatment of hyperpigmentation associated with the expression of the human tyrosinase gene.

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