RU2802418C2 - Antisense oligonucleotides of matrix metalloproteinase-1 - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to derivatives of peptide nucleic acids (PNA) targeting human matrix metalloproteinase-1 (MMP-1) pre-mRNA and their use for the treatment of skin aging conditions associated with human MMP-1 protein. A PNA derivative of formula I or a pharmaceutically acceptable salt thereof for inducing exon skipping in human MMP-1 pre-mRNA are proposed. A PNA derivative has at least a 14-dimensional complementary overlap with a 16-dimensional pre-mRNA sequence [(5'→ 3') CAUAUAUGGUGAGUAU] in human MMP-1 pre-mRNA. Also a pharmaceutical composition and a cosmetic composition for reducing skin aging associated with human MMP-1, and a method of reducing skin aging are proposed.

EFFECT: PNA derivative has good cellular or membrane permeability, can be locally administered as a "naked" oligonucleotide to induce MMP-1 "exon 5" skipping in the skin, improve skin condition and prevent its aging.

6 cl, 13 dwg, 8 ex

Description

TECHNICAL FIELD

This invention relates to peptide nucleic acid derivatives that additionally specifically target human matrix metalloproteinase-1 pre-mRNA to improve skin aging through matrix metalloproteinase-1.

PREREQUISITES FOR CREATION OF THE INVENTION

Skin aging has received significant attention because the signs of aging are most visible on the skin. Since the prevention and treatment of skin aging is very important for the quality of life, not only older people, but also young people are interested in appropriate healthy food, cosmetics, medicines, medical supplies, and so on. There are two main skin aging processes. One is aging caused by internal factors or natural aging, which accompanies aging, and the other is aging caused by external factors, which is caused by exogenous origin such as sun exposure, smoking and malnutrition.

Skin exposure to ultraviolet irradiation accelerates the expression of matrix metalloproteinase-1 (MMP-1), leading to accelerated breakdown of collagen, the main structural component of the dermis. In addition, smoking induces matrix metalloproteinase-1 mRNA in the skin, which leads to the same results as ultraviolet irradiation of the skin [J. Cosmetic Dermatology vol 6, 40-50 (2007)].

Collagen is the main structural protein in the extracellular space in various connective tissues such as skin, blood vessel, bone, tooth and muscle. Since collagen is responsible for supporting most tissues and cell structure, its breakdown and deformation can greatly affect skin aging [J. Pathol, vol 211, 241-251 (2007)].

Matrix metalloproteinases are enzymes that are secreted from fibroblast, keratinocyte, etc., which are capable of destroying all types of extracellular matrices (ECM) and basement membranes (BM). More than 26 types of matrix metalloproteinases have been identified, such as MMP-1 (interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (Matrilysin), MMP-8 (neutrophil collagenase) and MMP-12 ( metalloelastase) [J. Biol. Chem. vol 277, 451-454 (2002); J.Matrix. Biol, vol 15, 519-526 (1997)].

Reactive oxygen species induced by ultraviolet irradiation and smoking have been known to be due to the overexpression of matrix metalloproteinase-1. To treat skin aging in some sense, antioxidants and functional foods have been developed for antioxidant action, such as vitamin A (retinol), vitamin C (ascorbic acid), vitamin E (tocopherol), carotenoid, flavonoid, green tea and selenium, and currently Currently, the mechanism of their action is being studied [Int. J. Food Sci. Technol. vol 73, 989-996 (2005); Cor. J. Aesthet. Cosmetol. vol 11, 649-654 (2013); Int. J. Mol. Med. vol 38, 357-363 (2016)].

Considering the importance of metalloproteinase-1 in skin aging, it would be very interesting and necessary to develop pharmaceuticals or cosmetics based on the metalloproteinase-1 expression mechanism, which can improve and prevent the condition of skin aging.

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 schematically shown below. Exons and introns are numbered, as illustrated in the figure below:

Pre-mRNA splicing: Pre-mRNA is processed into mRNA after removal of introns by a series of complex reactions collectively called “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 the “splicosome E complex” (i.e., the early splicosome complex) between pre-mRNA and splicing adapter factors. In the "splicosome E complex", U1 binds to the exon N-intron N junction region, and U2AF ³⁵ binds to the N intron-exon junction region (N+1). Thus, exon/intron or intron/exon junction regions are critical for the formation of the early splicosome complex. The "splicosome E complex" is converted into the "splicosome A complex" upon additional complexation with U2. The "splicosome A complex" undergoes a series of complex reactions to remove or splice an intron to join 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-template ribonucleic acid) in the nucleus. Pre-mRNA introns are enzymatically spliced to produce mRNA (template ribonucleic acid), which is then translocated into the cytoplasm. In the cytoplasm, the translation structure 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 manner (i.e. complementary) is called an antisense oligonucleotide (ASO).

If ASO binds tightly to mRNA in the cytoplasm, for example, ASO can inhibit ribosomal protein synthesis from the mRNA. ASO is required 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 the splicing of pre-mRNA into mRNA. ASO is required 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 called "splice variant(s)" and encode protein(s) different from the protein encoded by full-length mRNA.

In principle, splicing can be disrupted by inhibiting the formation of the “splicosome E complex.” If ASO binds tightly to the exon-intron junction region (5'→3'), i.e. "5' splice site", then the ASO blocks the formation of a complex between the pre-mRNA and the U1 factor, and therefore the formation of the "splicosome E complex". Likewise, the "splicosome E complex" cannot form if the ASO binds tightly to the junction region (5'→3') intron-exon, i.e. "3' splice site".

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

Non-naturally occurring oligonucleotides: DNA or RNA oligonucleotides are susceptible to degradation by endogenous nucleases, limiting their therapeutic value. Currently, many types of oligonucleotides of non-natural origin (not found in nature) have been developed and intensively studied. [Clin. Exp.Pharmacol. Physiol, vol 33, 533-540 (2006)]. Some of them show long-term metabolic stability compared to DIC and RNA. Below are the chemical structures for several typical non-naturally occurring oligonucleotides. Such oligonucleotides bind predictably to complementary nucleic acid, just as DNA or RNA does.

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

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

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 therapeutic use in vivo.

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 antisense drug candidates have not been successfully developed, in part due to poor cell penetration of PTO. 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 toxicities, including prolonged clotting time, complement activation, tubular nephropathy, Kupffer cell activation, and immune stimulation including splenomegaly, lymphoid hyperplasia, and 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 liver or kidney involvement. Mipomersen is a PTO analogue 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 analogue of PTO, which inhibits the synthesis of protein tyrosine phosphatase 1B (PTP1B), and has been found to show therapeutic activity in patients with type 2 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 phosphorodiamidate morpholino oligonucleotide (PMO), the phosphate backbone and 2-deoxyribose of 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, the FMO backbone is not charged. Thus, the binding between FMO and mRNA does not have electrostatic repulsion between the backbones and is, as a rule, stronger than that between DNA and mRNA. Because FMO is structurally very different from DNA, FMO will not be recognized by the liver transporter(s) that recognize DNA or RNA. However, 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 complementary nucleic acid. [Nature (London) vol 365, 566-568 (1992)]. When bound to a complementary nucleic acid, the N-terminus of PNA is considered equivalent to the "5' end" of DNA or RNA and the C-terminus of PNA is considered equivalent to the "3' end" of DNA or RNA.

Like the FMO, the PNA backbone is uncharged. Thus, the binding between PNA and RNA is, as a rule, stronger than the binding between DNA and RNA. Because PNA is markedly different from DNA in chemical structure, PNA will not be recognized by the hepatic DNA-recognizing transporter(s) and will exhibit 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 membrane permeability of PNA; PNA was made highly membrane permeable to mammalian cells by introducing modified nucleic acid bases with a cationic lipid or its equivalent covalently attached to it. The chemical structures of such modified nucleic acid bases are given below. Such modified nucleobases of cytosine, adenine and guanine have been found to hybridize predictably and complementarily with guanine, thymine and cytosine, respectively. [PCT Appl. No. PCT/KR2009/001256; EP2268607; US8680253].

The incorporation of such modified nucleic acid bases into PNA has similarities to lipofection situations. In lipofection, the phosphate backbone oligonucleotide molecules are hidden by cationic lipid molecules such as lipofectamine, and such lipofectamine/oligonucleotide complexes tend to penetrate the membrane quite easily compared to naked oligonucleotide molecules.

It was found that in addition to good membrane permeability, these PNA derivatives have an ultra-strong affinity for complementary nucleic acid. For example, the introduction of 4 to 5 modified nucleic bases into 11-13-mer PNA derivatives easily gave an increase in T _m of 20°C or higher during duplex formation with complementary DNA. Such PNA derivatives are very sensitive to base pair mispairing. Base pair mispairing resulted in a loss of T _m ranging from 11 to 22°C depending on the type of base modified as well as the PNA sequence.

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). RISC then binds to a specific portion of the mRNA that is complementary to the antisense strand of the siRNA. The mRNA that has formed a complex with RISC undergoes cleavage. Thus, siRNA catalytically induces the cleavage of its and target mRNA, and consequently suppresses protein expression by the mRNA. RISC does not always bind to the complete complementary sequence within the target mRNA, raising concerns about off-targeting siRNA druggability. Like other classes of DNA or RNA backbone oligonucleotide, siRNA has poor cell permeability and therefore tends to exhibit poor therapeutic activity in vitro or in vivo unless properly prepared or chemically modified to obtain good membrane permeability.

Matrix metalloproteinase-1 siRNA: MMP-1 siRNA targeting the 19-mer RNA sequence in human MMP-1 mRNA has been reported to inhibit MMP-1 mRNA and protein expression in human chondrosarcoma after lipofection at 100 nM [J. Orthop.Res. vol 23, 1467-1474 (2005)]. This result may be useful in studying cancer metastasis associated with MMP-1 and developing cancer drugs.

DESCRIPTION OF THE INVENTION

Problem to be solved

Antioxidants and antioxidant functional foods have been developed to treat skin aging with controlled efficacy and the mechanism of action is currently being studied. It has been reported that although MMP-1-linked siRNA inhibits MMP-1 mRNA and protein expression in vitro, siRNAs are too expensive to produce and they must be delivered to the skin via transdermal administration to properly meet product guidelines. application. Therefore, considering the importance of metalloproteinase-1 in skin aging, it is very interesting and necessary to develop pharmaceuticals and cosmetics based on the expression mechanism of metalloproteinase-1, which can improve the condition of the skin and prevent its aging.

Solution

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

Where

n - integer from 10 to 21;

the compound of formula I has at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence [(5'→3') CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA;

a compound of formula I is fully complementary to human MMP-1 pre-mRNA or partially complementary to human MMP-1 pre-mRNA with one or two mismatches;

S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n independently represent a hydrogen atom, a deuterium atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group;

X and Y independently represent a hydrogen atom, a deuterium atom, 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 alkyloxy group, substituted or unsubstituted aryloxy 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 alkylaminothiocarbonyl group, substituted or unsubstituted arylaminothiocarbonyl group, substituted or unsubstituted alkyloxythiocarbonyl group, substituted or unsubstituted aryloxythiocarbonyl group, substituted or unsubstituted alkylsulfonyl group, substituted or unsubstituted arylsulfonyl group, substituted or an unsubstituted alkylphosphonyl group or a substituted or unsubstituted arylphosphonyl group.

Z represents a hydrogen atom, a deuterium 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 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; And

at least four of B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from non-naturally occurring nucleobases with a substituted or unsubstituted amino group covalently bonded to the nucleic base residue.

The compound of formula I induces skipping of “exon 5” in human MMP-1 pre-mRNA, produces splice variant(s) of human MMP-1 mRNA lacking “exon 5”, and is therefore suitable for inhibiting functional activity gene that reads human MMP-1 pre-mRNA.

The condition that "n is an integer between 10 and 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 the naturally occurring nucleic acid bases or non-naturally occurring nucleic acid bases in the PNA derivative of Formula I are exemplified in FIGS. 1a-1c. Natural (i.e., naturally occurring) nucleic acid bases or non-naturally occurring (i.e., not naturally occurring) nucleic acid bases described in the present invention include, but are not limited to, the nucleic acid bases shown in FIG. 1a-1c. The provision of such non-naturally occurring nucleic acid bases is illustrative of the variety of available nucleic acid bases, and therefore should not be construed to limit 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 arylsulfonyl group, and a substituted or unsubstituted alkylphosphonyl group or arylphosphonyl group. Fig. 2d gives examples for a substituted or unsubstituted alkyloxycarbonyl group or aryloxycarbonyl group, a substituted or unsubstituted alkylaminocarbonyl group or an arylaminocarbonyl group. In fig. 2e provides examples for 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 suitable substituents and therefore should not be construed to limit the scope of the present invention. One of ordinary skill in the art would readily recognize that the oligonucleotide sequence is the key factor for sequence-specific binding of the oligonucleotide to the target pre-mRNA sequence over 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 that is too high to be reliably determined in an aqueous buffer. The PNA compound of formula I gives high T _m values with complementary DNAs of shorter length.

The compound of formula I binds complementarily to the 5' splice site of "exon 5" of human MMP-1 pre-mRNA. [NCBI Reference Sequence: NG_011740]. The 16-mer sequence [(5'→3') CAUAUAUGGUGAGUAU] covers the junction region of “exon 5” and “intron 5” in human MMP-1 pre-mRNA, and consists of an 8-mer from “exon 5” and an 8-mer from "intron 5". Thus, a 16-mer pre-mRNA sequence may be generally denoted as [(5'→3') CAUAUAUG |gugaguau], where the exon and intron sequence are designated by "uppercase" and "lowercase" letters, respectively, and the exon-intron junction region is expressed by " |". The traditional designation for pre-mRNA is further illustrated by the 30-mer sequence [(5'→3') UCCAAGCCAUAUAUG |gugaguauggggaaa] spanning the junction region of “exon 5” and “intron 5” in human MMP-1 pre-mRNA.

The compound of formula I binds tightly to the target 5' splice site of human MMP-1 pre-mRNA read from the human MMP-1 gene and prevents the formation of an “early splicosome complex” to produce splice variant(s) of MMP-1 mRNA that do not contain (their) "exon 5" (exon 5 skipping).

The strong affinity of the RNA allows a compound of Formula I to cause MMP-1 "exon 5" skipping even if the PNA derivative has one or two mismatches to the target 5' splice site in the MMP-1 pre-mRNA. Likewise, a Formula I PNA derivative can still induce MMP-1 "exon 5" skipping in a mutant MMP-1 pre-mRNA possessing one or two single nucleotide polymorphisms (SNPs) at the target splice site.

The compound of formula I has good cell permeability and can be easily delivered into the cell as a naked oligonucleotide, as exemplified by the prior art [PCT/KR2009/001256]. Thus, the compound described in the present invention induces "exon 5" skipping in MMP-1 pre-mRNA and produces splice variant(s) of MMP-1 mRNA lacking MMP-1 “exon 5” in cells treated with a compound of formula I as a naked oligonucleotide. The compound of Formula I does not require any means or composition for delivery into the cell to potently induce target exon skipping in cells. The compound of formula I readily induces MMP-1 exon 5 skipping in cells treated with the compound described herein 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 MMP-1 exon 5 skipping in the skin. The compound of formula I does not require a dosage form to enhance 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 cosolvent and administered topically or transdermally at a sub-picomolar concentration to induce the desired therapeutic or biological activity in the target skin. The compound described in the present invention does not need to be administered intensively or aggressively to produce topical therapeutic activity. However, the PNA derivative of formula I can be formulated with cosmetic ingredients or adjuvants as a topical cream or lotion. Such a topical cosmetic cream or lotion may be suitable for treating skin aging.

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

The PNA derivative of Formula I can be formulated in a variety of ways, including, but not limited to, injection, nasal spray, transdermal patch, and the like. In addition, the PNA derivative of Formula I may be administered to a patient at a therapeutically effective dose, and the dosage of administration may vary depending on the indication, route of administration, dosage schedule, conditions or situations of the patient, and so on.

The compound of formula I can 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 a patient together 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.

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

Where

n - integer from 10 to 21;

the compound of formula I has at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence [(5'→3') CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA;

a compound of formula I is fully complementary to human MMP-1 pre-mRNA or partially complementary to human MMP-1 pre-mRNA with one or two mismatches;

S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n independently denote a hydrogen atom, a deuterium atom;

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

Z represents 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 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;

at least four of B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from the non-naturally occurring nucleic acid 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 hydrogen and a substituted or unsubstituted alkyl group;

L ₁ , L ₂ and L ₃ are a covalent linker, represented by formula V, covalently connecting a basic amino group to a nucleic base residue:

Where

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

Q ₂ , Q ₃ , ..., and Q _m-1 are 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 - an integer from 1 to 15.

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

n - integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence [(5'→3') CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA;

the compound of formula I is completely complementary to human MMP-1 pre-mRNA;

S ₁ , S ₂ , ..., 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 acyl group, or a substituted or unsubstituted alkyloxycarbonyl group;

Z represents a substituted or unsubstituted amino group;

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;

at least five of B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from the non-naturally occurring nucleic acid bases represented by formula II, formula III or formula IV;

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

Q ₁ and Q _m represent a methylene group and Q _m directly binds to the basic amino group;

Q ₂ , Q ₃ , ... and Q _m-1 are independently selected from a methylene group and 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 - integer from 11 to 16;

the compound of formula I has at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence [(5'→3') CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA;

the compound of formula I is completely complementary to human MMP-1 pre-mRNA;

S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n represent a hydrogen atom; X represents a hydrogen atom;

Y represents a substituted or unsubstituted alkyloxycarbonyl group;

Z represents a substituted or unsubstituted amino group;

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;

at least five of B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from the non-naturally occurring nucleic acid 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 a -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -group, -CH ₂ -O-(CH2) 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 connected to the main amino group.

Of interest is a PNA derivative of formula I, which is the compound below (ACO 1, antisense oligonucleotide 1), or a pharmaceutically acceptable salt thereof:

(N→C) Fethoc-TA(6)C-TCA(6)-CC(1O2)A(6)-TA(6)TA(6) T-NH ₂ ,

Where

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

C(pOq) and A(p) are PNA monomers with a non-naturally occurring nucleic acid base represented by formula VI and formula VII, respectively;

Where

p and q are integers; and p is 1 or 6 and q is 2 in ACO 1; And,

"Fethoc-" is an abbreviation for "[2-(9-fluorenyl)ethyl-1-oxy]carbonyl group" and "-NH ₂ " for the 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 considered to be a PNA monomer with a “modified cytosine” due to its hybridization to “guanine”. A(p) and A(pOq) are taken to be PNA monomers with “modified adenine” due to their hybridization into “thymine”.

To illustrate the abbreviations used for such PNA derivatives, the chemical structure of ACO 1 is shown in FIG. 4.

ACO 1 is equivalent to the DNA sequence "(5'→3') TAC-TCA-CCA-TAT-AT" for complementary binding to pre-mRNA. The 14-mer PNA has a 14-mer complementary overlap with the 14-mer sequence designated "bold" and "underlined" in the 30-mer RNA sequence [(5'→3')

UCCAAGCCAUAUAUG |gugaguauggggaaa], covering the junction region of “exon 5” and “intron 5” in human MMP-1 pre-mRNA.

One aspect of the present invention is a method of treating diseases or conditions associated with transcription of the human MMP-1 gene, comprising administering to a patient a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

One aspect of the present invention is a method of treating skin aging comprising administering to a patient a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

One aspect of the present invention is a pharmaceutical composition for the treatment of diseases or conditions associated with transcription of the human MMP-1 gene, comprising a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

One aspect of the present invention is a cosmetic composition for the treatment of diseases or conditions associated with transcription of the human MMP-1 gene, comprising a peptide nucleic acid derivative in accordance with the present invention or a pharmaceutically acceptable salt thereof.

One aspect of the present invention is a pharmaceutical composition for treating skin aging comprising a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

One aspect of the present invention is a cosmetic composition for treating skin aging comprising a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

Diseases or conditions associated with transcription of the human MMP-1 gene can be treated by administering a PNA derivative of Formula I or a pharmaceutically acceptable salt thereof.

Diseases or conditions associated with skin aging can be treated by administering a PNA derivative of Formula I or a pharmaceutically acceptable salt thereof.

Brief explanation of figures

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

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

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

Fig. 4. Chemical structure of "ASO 1".

Fig. 5. Chemical structures of Fmoc-PNA monomers used for the synthesis of PNA derivatives of the present invention.

Fig. 6a-6b. C ₁₈ -reverse phase HPLC chromatograms of "ACO 1" before and after HPLC purification, respectively.

Fig. 7. ESI-TOF mass spectrum of “ACO 1” purified by C ₁₈ -RP preparative HPLC.

Fig. 8. Inhibition of the formation of MMP-1 mRNA using "ASO 1" in HDF (HDF - human dermal fibroblasts, human dermal fibroblasts) (quantitative PCR with real-time detection).

Fig. 9a-9b. Inhibition of MMP-1 protein expression by "ACO 1" in HDF (Western blot and graph of changes in protein expression levels).

Fig. 10a-10b. Enhancement of collagen protein expression by "ACO 1" in HDF (Western blot and protein expression levels graph).

Fig. 11a-11b. Inhibition of MMP-1 protein expression by "ASO 1" in extracellular fluid (Western blot and graph of changes in protein expression levels).

Fig. 12a-12b. Increased expression of collagen protein by "ASO 1" in extracellular fluid (Western blot and graph of changes in protein expression levels).

Fig. 13. Inhibition of MMP-1 protein expression using "ASO 1" in extracellular fluid (ELISA).

Best Mode for Carrying Out the Invention

General methods for preparing 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 changes. 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. To synthesize the PNA derivatives of the present invention, specific Fmoc-PNA monomers with a modified nucleic acid base and Fmoc-PNA monomers with a naturally occurring nucleic acid base were used. PNA oligomers were purified by _C18 reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

Scheme 1 illustrates the typical monomer elongation cycle adopted in the SPPS of this study, and the synthetic details are given below. However, to one skilled in the art, there are many minor changes that are obviously possible when such SPPS reactions are carried out efficiently on an automated peptide synthesizer or a non-automated peptide synthesizer. Each reaction step in Scheme 1 is briefly summarized as follows.

[Scheme 1]

[DeFmoc] The resin was shaken in 1.5 ml of 20% piperidine/DMF for 7 min and the DeFmoc solution was filtered. The resin was washed for 30 sec each time successively 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 the solid support were immediately coupled to the Fmoc-PNA monomer.

[Binding to Fmoc-PNA Monomer] Free amines on a solid support were bound to Fmoc-PNA monomer as follows. 0.04 mmol PNA monomer, 0.05 mmol HBTU and 10 mmol DIEA were incubated for 2 min in 1 ml anhydrous DMF and added to the free amine resin. The resin solution was shaken for 1 hour and the reaction mixture was filtered. The resin was then washed for 30 sec 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. Nucleic base 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 number of changes to the Fmoc-PNA monomers to synthesize the PNA derivative of Formula I can readily be appreciated by one skilled in the art.

[Capping] After the coupling reaction, unreacted free amines were copied by shaking for 5 minutes into 1.5 ml capping solution (5% acetic anhydride and 6% 2,6-leuthidine in DMF). The capping solution was then filtered and washed for 30 sec successively each time with 1.5 ml MS, 1.5 ml DMF and 1.5 ml MS.

[Introduction of a "Fethoc-" group at the N-terminus] A "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, C20H17NO5, M.B.351.36] is given as follows.

[Resin Cleavage] PNA oligomers bound to the resin were cleaved from the resin by shaking for 3 hours in 1.5 ml of a cleavage solution (2.5% triisopropylsilane 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 reverse-phase HPLC filtration.

[HPLC Analysis and Purification] After cleavage of the resin, 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. 6a and 6b represent typical HPLC chromatograms for "ASO 1" before and after HPLC purification, respectively.

Synthetic examples for the PNA derivative of formula I

In order to complementarily target the 5' splice site of "exon 5" in human MMP-1 pre-mRNA, the PNA derivative of the present invention was prepared according to the synthesis procedures given above or with minor modifications. The provision of such PNA derivatives targeting human MMP-1 pre-mRNA is an example of Formula I PNA derivatives and should not be construed as limiting the scope of the present invention.

Table 1 shows a PNA derivative that complementarily targets the 5' splice site of "exon 5" in the human MMP-1 pre-mRNA read from the human MMP-1 gene [NCBI (National Center for Biotechnology Information) Standard/Reference Sequence: NG 011740] , as well as data on structural studies using mass spectrometry. The provision of a peptide nucleic acid derivative in Table 1 is an example of a PNA derivative of Formula I, and should not be construed to limit the scope of the present invention.

"ASO 1" has a 14-mer complementary overlap with the 14-mer sequence designated "bold" and "underlined" within the 30-mer RNA sequence [(5'→3')

UCCAAGCCAUAUAUG |gugaguauggggaaa], covering the junction region of “exon 5” and “intron 5” in human MMP-1 pre-mRNA. Thus, “ASO 1” has a 7-mer overlap with “exon 5” and a 7-mer overlap with “intron 5” within the human MMP-1 pre-mRNA.

Binding affinity "ACQ 1" for complementary DNA

The T _m values were determined using a spectrometer with UV-visible spectral regions as follows. A mixed solution of 4 µM PNA oligomer and 4 µM complementary 14-mer DNA in 4 ml of aqueous buffer (pH 7.16, 10 mM sodium phosphate, 100 mM NaCl) in a 15 ml polypropylene tube from Falcon was incubated at 90°C for a minute and slowly cooled to normal temperature. The solution was then transferred to a 3 ml quartz UV cuvette equipped with a sealed lid and T _m was measured at 260 nm on a UV-visible spectrophotometer (Agilent 8453). Absorbance changes at 260 nm were recorded as the cuvette temperature increased by either 0.5 or 1.0°C per minute. From the absorption versus temperature curve, the temperature showing the highest absorption intensity was read as the melting temperature T _m from PNA to DNA. 14-mer complementary DNA(s) for T _m measurements were purchased from Bioneer (www.bioneer.com, Dajeon, Republic of Korea) and used without further purification.

"ACO 1" showed a T _m value of 72.67°C for a duplex with 14-mer complementary DNA.

Examples of biological activities of PNA derivatives of formula I

The PNA derivatives of this invention were assessed in vitro for MMP-1 antisense activities in human dermal fibroblasts (HDF) using real-time quantitative polymerase chain reaction (RT qPCR) etc. .d.. The biological examples are provided as examples to illustrate the biological profiles of the PNA derivatives of formula I, and therefore should not be construed to limit the scope of the present invention.

Example 1. Inhibition of MMP-1 mRNA formation using "ACO 1" bHDF.

"ASO 1" was assessed by Western blotting for its ability to suppress the formation of MMP-1 mRNA in HDF, as described below.

[Cell Culture and ASO Treatment] HDF cells were maintained in fibroblast basal medium (ATCC PCS-201-030) supplemented with low serum fibroblast growth kit (ATCC PCS-201-041) and 1% streptomycin/penicillin, which was grown at 37°C and subject to 5% CO ₂ . HDF cells (3×10 ⁵ ), stabilized for 24 hours in a 60 mm culture dish, were incubated for 24 hours with “ACO 1” at 0 (negative control) and 100 aM to 1 μM.

[RNA Extraction and cDNA Synthesis] Total RNA was extracted using the RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions from ASO 1 treated cells and cDNA was prepared from 400 ng RNA using the PrimeScript 1-Strand cDNA Synthesis Kit ™ (Takara, Cat. No. 6110A). DEPC-treated water was added to a mixture of 400 ng RNA, 1 μL random hexamer, and 1 μL dNTP (deoxynucleoside triphosphate) (10 mM) in a PCR tube to a total volume of 10 μL, which was reacted at 65°C for 5 minutes. cDNA was synthesized by adding 10 μl of PrimeScript RTase to the reaction mixture and reacting at 30°C for 10 minutes and at 42°C for 60 minutes, sequentially.

[Quantitative real-time PCR] In order to evaluate the expression level of human MMP-1 mRNA, quantitative real-time PCR was performed on the synthesized cDNA using a linear degradable probe. A mixture of cDNA, linear degradable probe, IQ supermix (BioRad, Cat. No. 170-8862) and water without nuclease activity in a PCR tube was reacted using a CFX96 blind real-time method (BioRad) according to the cycling conditions specified as follows: 95°C for 3 min (primary denaturation) followed by 50 cycles of 10 sec at 95°C (denaturation), 30 sec at 60°C (annealing) and 30 sec at 72°C (polymerization). Fluorescence intensity was measured at the end of each cycle and the PCR result was assessed using a melting curve. After the threshold cycle (Ct) of each gene was standardized by GAPDH, the change in Ct was compared and analyzed.

[Reduction of MMP-1 mRNA by ASO] As can be seen from FIG. 8, compared with the control experiment, the amount of reduced MMP-1 mRNA was 65% with 1 μM "ACO 1" treatment and from 10 to 15% with 100 aM to 10 nM μM "ACO 1" treatment, respectively.

Example 2: Inhibition of MMP-1 protein expression by "ASO 1" in HDF.

"ASO 1" was assessed by Western blotting for its ability to suppress MMP-1 protein expression in HDF, as described below.

[Western blotting] HDF cells were grown as in Example 1 and 48 hours later washed 2 times with cold PBS (phosphate buffered saline) and dissolved in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS, protease inhibitor. Protein was quantified with BCA solution (Thermo, Cat. No. 23225) and purified by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE Gel). The protein was transferred to a PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was tested with anti-MMP-1 (SantaCruz, Cat. No. 58377) and anti-P-actin (Sigma, Cat. No. A3854) as the primary antibody and goat anti-mouse antibody (CST, Cat. No.) as the secondary antibody. 7076V). SuperSignal™ West Pico (PierAce, USA) was used to detect the chemiluminescent signal and the signal intensity was measured using a Gel Doc system (ATTO). Based on the Western blot results of each band, the relative expression levels of MMP-1 were quantified using the Image-J program.

[Inhibition of MMP-1 Protein Expression by ASO] As shown in FIG. 9a and 9b, compared to the control experiment, the reduced magnitude of MMP-1 protein expression ranged from 20 to 50% at 100 aM to 1 μM "ACO 1" treatment. Thus, it was proven that "ASO 1" showed its ability to suppress the expression of MMP-1 protein in HDF.

Example 3. Increasing the expression of collagen protein using "ACO 1" b HDF.

"ACO 1" was assessed by Western blotting for its ability to upregulate the expression of type I collagen protein in HDF, associated with a decrease in MMP-1 protein expression, as described below.

[Western Blot] HDF cells were grown as in Example 1 and 48 hours later washed 2 times with cold PBS (phosphate buffered saline) and dissolved in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS, protease inhibitor. Protein was quantified with BCA solution (Thermo, Cat. No. 23225) and purified by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE Gel). The protein was transferred to a PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was tested with anti-MMP-1 (SantaCruz, Cat. No. 58377) and anti-P-actin (Sigma, Cat. No. A3854) as the primary antibody and rabbit anti-goat antibody (Santacruz, Cat. No. 2768). SuperSignal™ West Pico (PierAce, USA) was used to detect the chemiluminescent signal and the signal intensity was measured using a Gel Doc system (ATTO). Based on the Western blot results of each band, the relative expression levels of MMP-1 were quantified using the Image-J program.

[Increasing Type I Collagen Protein Expression by ASO] As shown in FIG. 10a and 10b, compared with the control experiment, the increased expression level of tier I collagen protein was more than 30% at 100 aM to 1 μM "ACO 1" treatment. Thus, it was proven that the “ACO 1”-induced decrease in MMP-1 protein expression showed its ability to increase the expression of type I collagen protein in HDF.

Example 4. Inhibition of MMP-1 protein expression using "ACO 1" in extracellular fluid (Western blotting)

The reduction in MMP-1 protein expression caused by "ASO 1" in the cell may, as a result, affect the level of MMP-1 protein expression secreted outside the cell. In this sense, "ASO 1" was evaluated by Western blotting for its ability to suppress the expression of MMP-1 protein in cell culture fluid 48 hours after treatment with "ASO 1" as described below.

[Western Blot] HDF cells were grown as in Example 1, and 48 hours later, the collected cell culture fluid was purified by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE Gel). The isolated protein was transferred to a PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was tested with anti-MMP-1 (SantaCruz, Cat. No. 58377) as the primary antibody and goat anti-mouse antibody (CST, Cat. No. 7076V) as the secondary antibody. SuperSignal™ West Pico (PierAce, USA) was used to detect the chemiluminescent signal and the signal intensity was measured using a Gel Doc system (ATTO). Based on the Western blot results of each band, the relative expression levels of MMP-1 were quantified using the Image-J program.

[Inhibition of MMP-1 protein expression by ASO] As shown in Figs. 11a and 11b, compared with the control experiment, the reduced magnitude of MMP-1 protein expression ranged from 10% to 60% at 100 pM to 1 μM "ASO 1" treatment "in the extracellular fluid. Thus, it was proven that "ASO 1" showed its ability to suppress the expression of MMP-1 protein in the extracellular fluid.

Example 5. Increasing the expression of collagen protein using "ASO 1" in extracellular fluid

"ASO 1" was assessed by Western blotting for its ability to activate the expression of type I collagen protein in the extracellular fluid, as described below.

[Western Blot] HDF cells were grown as in Example 1, and 48 hours later, the collected cell culture fluid was purified by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE Gel). The isolated protein was transferred to a PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was tested with anti-MMP-1 (SantaCruz, Cat. No. 58377) as the primary antibody and rabbit anti-goat antibody (Santacruz, Cat. No. 2768) as the secondary antibody. SuperSignal™ West Pico (PierAce, USA) was used to detect the chemiluminescent signal and the signal intensity was measured using a Gel Doc system (ATTO). Based on the Western blot results of each band, the relative expression levels of MMP-1 were quantified using the Image-J program.

[Increasing Type I Collagen Protein Expression by ASO] As shown in FIG. 12a and 12b, compared with the control experiment, the increased expression level of collagen protein was 20% at 1 pM and 80% at 1 μM "ACO 1" treatment, respectively. Thus, it was proven that the decrease in MMP-1 protein expression induced by “ACO 1” in HDF showed its ability to activate the expression of type I collagen protein in the extracellular fluid.

Example 6 Inhibition of MMP-1 protein expression using "ASO 1" in extracellular fluid (ELISA).

The reduction in MMP-1 protein expression caused by "ASO 1" in the cell may, as a result, affect the level of MMP-1 protein expression secreted outside the cell. In this sense, "ACO 1" was evaluated by enzyme linked immunosorbent assay (ELISA) for its ability to suppress the expression of MMP-1 protein in cell culture fluid 48 hours after treatment with "ASO 1", as described below.

[ELISA] HDF cells were grown as in Example 1, and 48 hours later the collected cell culture fluid was assessed for MMP-1 expression by absorption (Sunrise, TEC AN) with a human MMP-1 ELISA kit (abeam, Cat. No. ab 100603) according to the manufacturer's instructions.

[Inhibition of MMP-1 Protein Expression by ASO] As shown in FIG. 13, compared with the control experiment, the magnitude of the reduced level of MMP-1 protein expression ranged from 15% to 30% at 100 pM to 10 nM and 50% at 1 μM "ASO 1" treatment, respectively. Thus, it was proven that "ASO 1" showed its ability to suppress the expression of MMP-1 protein in the extracellular fluid.

Example 7 Preparation of a compound of formula I containing topical serum (wt/wt%)

A compound of formula I, eg "ASO 1", was formulated as a serum for topical use in patients. Serum for topical application was prepared as described below. Given that there are many variations of possible Topical Serum, this preparation should be considered as an example and should not be interpreted to limit the scope of the present invention.

In a separate beaker, the mixed substances of part A and part B were dissolved at 25°C, respectively. Part A and Part B were mixed and emulsified using a homogenizer at 3600 rpm at 25°C for 5 minutes. The emulsified part C was filtered through a 50 mesh filter and the filtrate was added to the mixture of parts A and B. The resulting mixture was emulsified using a homogenizer at 3600 rpm at 80°C for 5 minutes. After adding part D to the mixture of parts A, B and C, the resulting mixture was emulsified using a homogenizer at 2500 rpm at 25°C for 3 minutes. Finally, ensure uniform dispersion and complete destruction of foam.

Example 8 Preparation of a topical cream containing a compound of formula I (wt/wt%)

A compound of formula I, eg "ASO 1", was formulated as a topical cream for patients. The topical cream was prepared as described below. Given that there are many possible options for a topical cream, this preparation should be considered as an example and should not be interpreted as limiting the scope of the present invention.

In a separate beaker, the substances of part A were dissolved at 80°C and part B at 85°C, respectively. Part A and Part B were mixed and emulsified using a homogenizer at 3600 rpm at 80°C for 5 minutes. After adding parts C and D to the mixture of parts A and B, the resulting mixture was emulsified using a homogenizer at 3600 rpm at 80°C for 5 minutes. After adding part E to the mixture of parts A, B, C and D at 35°C, the resulting mixture was emulsified using a homogenizer at 3600 rpm at 35°C for 3 minutes. Finally, ensure uniform dispersion and complete destruction of foam at 25°C.

Claims (44)

1. A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof, for inducing exon skipping in human matrix metalloproteinase-1 (MMP-1) pre-mRNA: Where n - integer from 14 to 21; a compound of formula I has at least a 14-mer complementary overlap with a 16-mer pre-mRNA sequence in human MMP-1 pre-mRNA; a compound of formula I is fully complementary to human MMP-1 pre-mRNA or partially complementary to human MMP-1 pre-mRNA with one mismatch; S ₁ , S ₂ , ..., S _n-1 , S _n , T ₁ , T ₂ , ..., T _n-1 and T _n represent a hydrogen atom; X represents a hydrogen atom; Y represents a substituted or unsubstituted alkyloxycarbonyl group, wherein the substituted or unsubstituted alkyloxycarbonyl group is selected from: Z denotes an amino group; B ₁ , B ₂ , ..., B _n-1 and B _n are independently selected from naturally occurring nucleic acid bases, including adenine, thymine, guanine, cytosine and uracil, and non-naturally occurring nucleic acid bases; wherein the number of non-naturally occurring nucleic acid bases is 6 and the non-naturally occurring nucleic acid bases are independently selected from Formula II, Formula III or Formula IV: Where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ represent a hydrogen atom; L ₁ , L ₂ and L ₃ are a covalent linker, represented by formula V, covalently connecting a basic amino group to a nucleic base residue: Where Q ₁ and Q _m represent a methylene group (-CH ₂ -) and Q _m directly connects the main amino group; Q ₂ , Q ₃ , ... and Q _m-1 are independently selected from a methylene group and an oxygen atom; and m is an integer from 1 to 15. 2. A peptide nucleic acid derivative according to claim 1 or a pharmaceutical salt thereof, where n - integer from 14 to 18; a compound of formula I has at least a 14-mer complementary overlap with a 16-mer pre-mRNA sequence in human MMP-1 pre-mRNA; the compound of formula I is fully complementary to human MMP-1 pre-mRNA; 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 represents a substituted or unsubstituted alkyloxycarbonyl group; Z denotes an amino group; 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-natural nucleic bases in which the number of non-natural nucleic bases is 6 and bases of non-natural origin are independently selected from 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 stands for -(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 binds to the main 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 ) _b -, -(CH ₂ ) ₇ - and -(CH ₂ ) ₈ - with the right end directly binds to the main amino group. 3. The peptide nucleic acid derivative according to claim 2, which is the peptide nucleic acid derivative given below or a pharmaceutically acceptable salt thereof: (N→C) Fethoc-TA(6)C-TCA(6)-CC(1O2)A(6)-TA(6)TA(6)T-NH ₂ , Where A, T and C are peptide nucleic acid monomers with the natural nucleic base adenine, thymine and cytosine, respectively; C(pOq) and A(p) are peptide nucleic acid monomers with a non-naturally occurring nucleic base represented by Formula VI and Formula VII, respectively; Where p and q are integers and p is 1 or 6 and q is 2; And "Fethoc-" is an abbreviation for "[2-(9-fluorenyl)ethyl-1-oxy]carbonyl group". 4. A method for reducing skin aging associated with increased expression of MMP-1, comprising administering to the patient a peptide nucleic acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof. 5. A pharmaceutical composition for reducing skin aging associated with increased expression of MMP-1, comprising a peptide nucleic acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant. 6. A cosmetic composition for reducing skin aging associated with increased expression of MMP-1, comprising a peptide nucleic acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof and cosmetic ingredients.

Images