MATRIX METALLOPROTEINASE-1 ANTISENSE OLIGONUCLEOTIDES

Abstract

A method to treat diseases or conditions associated with the human MMP-1 gene transcription involving administration of the peptide nucleic acid derivative according to claim 1 to a subject. The present invention provides the peptide nucleic acid derivative according to claim 1 which targets 5′ splice site of the human MMP-1 pre-mRNA “exon 5”. The peptide nucleic acid derivatives in the present invention strongly induce splice variants of the human MMP-1 mRNA in cell and are very useful to treat conditions or diseases of skin aging associated with the human MMP-1 protein.

Claims

1. A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof: ##STR00010## wherein, n is an integer between 10 and 21; the compound of Formula I possesses at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence of [(5′.fwdarw.3′) CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA; the compound of Formula I is fully complementary to the human MMP-1 pre-mRNA, or partially complementary to the human MMP-1 pre-mRNA with one or two mismatches; S.sub.1, S.sub.2, . . . , S.sub.n-1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n-1, and T.sub.n independently represent hydrido, deuterido, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical; X and Y independently represent hydrido, deuterido, formyl [H—C(═O)—], aminocarbonyl [NH.sub.2—C(═O)—], aminothiocarbonyl [NH.sub.2—C(═S)—], substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, substituted or non-substituted aryloxycarbonyl, substituted or non-substituted alkylaminocarbonyl, substituted or non-substituted arylaminocarbonyl, substituted or non-substituted alkylaminothiocarbonyl, substituted or non-substituted arylaminothiocarbonyl, substituted or non-substituted alkyloxythiocarbonyl, substituted or non-substituted aryloxythiocarbonyl, substituted or non-substituted alkylsulfonyl, substituted or non-substituted arylsulfonyl, substituted or non-substituted alkylphosphonyl, or substituted or non-substituted arylphosphonyl radical; Z represents hydrido, deuterido, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical; B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and, at least four of B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from unnatural nucleobases with a substituted or non-substituted amino radical covalently linked to the nucleobase moiety.

2. The peptide nucleic acid derivative according to claim 1, or a pharmaceutical salt thereof: wherein, n is an integer between 10 and 21; the compound of Formula I possesses at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence of [(5′.fwdarw.3′) CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA; the compound of Formula I is fully complementary to the human MMP-1 pre-mRNA, or partially complementary to the human MMP-1 pre-mRNA with one or two mismatches; S.sub.1, S.sub.2, . . . , S.sub.n-1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n-1, and T.sub.n independently represent hydrido, deuterido radical; X and Y independently represent hydrido, deuterido, formyl [H—C(═O)—], aminocarbonyl [NH.sub.2—C(═O)—], aminothiocarbonyl [NH.sub.2—C(═S)—], substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, substituted or non-substituted aryloxycarbonyl, substituted or non-substituted alkylaminocarbonyl, substituted or non-substituted arylaminocarbonyl, substituted or non-substituted alkylaminothiocarbonyl, substituted or non-substituted arylaminothiocarbonyl, substituted or non-substituted alkyloxythiocarbonyl, substituted or non-substituted aryloxythiocarbonyl, substituted or non-substituted alkylsulfonyl, substituted or non-substituted arylsulfonyl, substituted or non-substituted alkylphosphonyl, or substituted or non-substituted arylphosphonyl radical; Z represents-hydrido, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, or substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least four of B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV: ##STR00011## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently selected from hydrido and substituted or non-substituted alkyl radical; L.sub.1, L.sub.2 and L.sub.3 are a covalent linker represented by Formula V covalently linking the basic amino group to the nucleobase moiety: ##STR00012## wherein, Q.sub.1 and Q.sub.m are substituted or non-substituted methylene (—CH.sub.2—) radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m-1 are independently selected from substituted or non-substituted methylene, oxygen (—O—), sulfur (—S—), and substituted or non-substituted amino radical [—N(H)—, or —N(substituent)-]; and, m is an integer between 1 and 15.

3. The peptide nucleic acid derivative according to claim 2, or a pharmaceutical salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence of [(5′.fwdarw.3′) CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA; the compound of Formula I is fully complementary to the human MMP-1 pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n-1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n-1, and T.sub.n are hydrido radical; X and Y independently represent hydrido, substituted or non-substituted alkylacyl, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least five of B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are hydrido radical; Q.sub.1 and Q.sub.m are methylene radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m-1 are independently selected from methylene and oxygen radical; and, m is an integer between 1 and 9.

4. The peptide nucleic acid derivative according to claim 3, or a pharmaceutical salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 16-mer pre-mRNA sequence of [(5′.fwdarw.3′) CAUAUAUGGUGAGUAU] in the human MMP-1 pre-mRNA; the compound of Formula I is fully complementary to the human MMP-1 pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n-1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n-1, and T.sub.n are hydrido radical; X is hydrido radical; Y represents substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least five of B.sub.1, B.sub.2, . . . , B.sub.n-1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are hydrido radical; L.sub.1 represents —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—, —CH.sub.2—O—(CH.sub.2).sub.2, —CH.sub.2—O—(CH.sub.2).sub.3—, —CH.sub.2—O—(CH.sub.2).sub.4—, or —CH.sub.2—O—(CH.sub.2).sub.5— with the right end is directly linked to the basic amino group; and, L.sub.2 and L.sub.3 are independently selected from —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—O—(CH.sub.2).sub.2—, —(CH.sub.2).sub.2—O—(CH.sub.2).sub.3—, —(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.6—, —(CH.sub.2).sub.7—, and —(CH.sub.2).sub.8— with the right end is directly linked to the basic amino group.

5. The peptide nucleic acid derivative according to claim 4, which is a peptide nucleic acid derivative provided below, or a pharmaceutically acceptable salt thereof: (N.fwdarw.C) Fethoc-TA(6)C-TCA(6)-CC(102)A(6)-TA(6)T-A(6)T-NH.sub.2 wherein, A, T, and C are monomers of peptide nucleic acid with a natural nucleobase of adenine, thymine, and cytosine, respectively; C(pOq) and A(p) are monomers of peptide nucleic acid with an unnatural nucleobase represented by Formula VI and Formula VII, respectively; ##STR00013##  wherein, p and q are integers and p is 1 or 6, and q is 2; and, “Fethoc-” is the abbreviation for “[2-(9-fluorenyl)ethyl-1-oxy]carbonyl”.

6. A method to treat diseases or conditions associated with the human MMP-1 gene transcription, comprising the administration of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof to a subject.

7. A method to treat skin aging, comprising the administration of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof to a subject.

8. A pharmaceutical composition for treating diseases or conditions associated with human MMP-1 gene transcription, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

9. A cosmetic composition for treating diseases or conditions associated with human MMP-1 gene transcription, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition for treating skin aging, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

11. A cosmetic composition for treating skin aging, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

Description

BRIEF EXPLANATION OF DRAWINGS

[0133] FIGS. 1a-1c. Examples of natural or unnatural (modified) nucleobases selectable for the peptide nucleic acid derivative of Formula I.

[0134] FIGS. 2a-2e. Examples of substituents selectable for the peptide nucleic acid derivative of Formula I.

[0135] FIG. 3. Chemical structures of PNA monomers with natural or modified nucleobase.

[0136] FIG. 4. Chemical structure of “ASO 1”.

[0137] FIG. 5. Chemical structures of Fmoc-PNA monomers used to synthesize the PNA derivatives of this invention.

[0138] FIGS. 6a-6b. C.sub.18-reverse phase HPLC chromatograms of “ASO 1” before and after HPLC purification, respectively.

[0139] FIG. 7. ESI-TOF mass spectrum of “ASO 1” purified by C.sub.18-RP prep HPLC.

[0140] FIG. 8. Inhibition of MMP-1 mRNA Formation by “ASO 1” in HDF (Real-Time qPCR).

[0141] FIG. 9a-9b. Inhibition of MMP-1 Protein Expression by “ASO 1” in HDF (Western Blot and Graph for Protein Expression Level Changes).

[0142] FIG. 10a-10b. Enhancement of Collagen Protein Expression by “ASO 1” in HDF (Western Blot and Graph for Protein Expression Level Changes).

[0143] FIG. 11a-11b. Inhibition of MMP-1 Protein Expression by “ASO 1” in extracellular fluid (Western Blot and Graph for Protein Expression Level Changes).

[0144] FIG. 12a-12b. Enhancement of Collagen Protein Expression by “ASO 1” in extracellular fluid (Western Blot and Graph for Protein Expression Level Changes).

[0145] FIG. 13. Inhibition of MMP-1 Protein Expression by “ASO 1” in extracellular fluid (ELISA).

[0146] FIG. 14. Mammalian pre-mRNA exons and introns.

[0147] FIG. 15. Pre-mRNA is processed into mRNA following deletion of introns by a series of complex reactions collectively called “splicing”.

[0148] FIG. 16. 3′ splice site and 5′ splice site.

BEST MODE FOR CARRYING OUT THE INVENTION

[0149] General Procedures for Preparation of PNA Oligomers

[0150] PNA oligomers were synthesized by solid phase peptide synthesis (SPPS) based on Fmoc-chemistry according to the method disclosed in the prior art [U.S. Pat. No. 6,133,444; WO96/40685] with minor but due modifications. The solid support employed in this study was H-Rink Amide-ChemMatrix purchased from PCAS BioMatrix Inc. (Quebec, Canada). Fmoc-PNA monomers with a modified nucleobase were synthesized as described in the prior art [PCT/KR 2009/001256] or with minor modifications. Such Fmoc-PNA monomers with a modified nucleobase and Fmoc-PNA monomers with a naturally occurring nucleobase were used to synthesize the PNA derivatives of the present invention. PNA oligomers were purified by C.sub.18-reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

[0151] Scheme 1 illustrates a typical monomer elongation cycle adopted in the SPPS of this study, and the synthetic details are provided as below. To a skilled person in the field, however, there are lots of minor variations obviously possible in effectively running such SPPS reactions on an automatic peptide synthesizer or manual peptide synthesizer. Each reaction step in Scheme 1 is briefly provided as follows.

##STR00008##

[0152] [DeFmoc] The resin was vortexed in 1.5 mL 20% piperidine/DMF for 7 min, and the DeFmoc solution was filtered off. The resin was washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The resulting free amines on the solid support were immediately subjected to coupling with an Fmoc-PNA monomer.

[0153] [Coupling with Fmoc-PNA Monomer] The free amines on the solid support were coupled with an Fmoc-PNA monomer as follows. 0.04 mmol of PNA monomer, 0.05 mmol HBTU, and 10 mmol DIEA were incubated for 2 min in 1 mL anhydrous DMF, and added to the resin with free amines. The resin solution was vortexed for 1 hour and the reaction medium was filtered off. Then the resin was washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The chemical structures of Fmoc-PNA monomers with a modified nucleobase used in this invention are provided in FIG. 6. The Fmoc-PNA monomers with a modified nucleobase are provided in FIG. 6 should be taken as examples, and therefore should not be taken to limit the scope of the present invention. A skilled person in the field may easily figure out a number of variations in Fmoc-PNA monomers to synthesize the PNA derivative of Formula I.

[0154] [Capping] Following the coupling reaction, the unreacted free amines were capped by shaking for 5 min in 1.5 mL capping solution (5% acetic anhydride and 6% 2,6-leutidine in DMF). Then the capping solution was filtered off and washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC.

[0155] [Introduction of “Fethoc-” Radical in N-Terminus] “Fethoc-” radical was introduced to the N-terminus by reacting the free amine on the resin with “Fethoc-OSu” under basic coupling conditions. The chemical structure of “Fethoc-OSu” [CAS No. 179337-69-0, C.sub.20H.sub.17NO.sub.5, MW 351.36] is provided as follows.

##STR00009##

[0156] [Cleavage from Resin] PNA oligomers bound to the resin were cleaved from the resin by shaking for 3 hours in 1.5 mL 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 for purification by reverse phase HPLC.

[0157] [HPLC Analysis and Purification] Following a cleavage from resin, the crude product of a PNA derivative was purified by C.sub.18-reverse phase HPLC eluting water/acetonitrile or water/methanol (gradient method) containing 0.1% TFA. FIGS. 6a and 6b are exemplary HPLC chromatograms for “ASO 1” before and after HPLC purification, respectively.

Synthetic Examples for PNA Derivative of Formula I

[0158] In order to complementarily target the 5′ splice site of “exon 5” in the human MMP-1 pre-mRNA, PNA derivatives of this invention were prepared according to the synthetic procedures provided above or with minor modifications. Provision of such PNA derivatives targeting the human MMP-1 pre-mRNA is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

TABLE-US-00001 TABLE 1 PNA derivative complementarily targeting the 5′ splice site of “exon 5” in the human MMP-1 pre-mRNA along with structural characterization data by mass spectrometry. PNA Exact Mass, m/z Example PNA Sequence (N .fwdarw. C) theor..sup.a obs..sup.b ASO 1 Fethoc-TA(6)C-TCA(6)- 4631.22 4631.22 CC(1O2)A(6)-TA(6)T- A(6)T-NH.sub.2 .sup.atheoretical exact mass, .sup.bobserved exact mass

[0159] Table 1 provides PNA derivative complementarily targeting the 5′ splice site of “exon 5” in the human MMP-1 pre-mRNA read out from the human MMP-1 gene [NCBI Reference Sequence: NG_011740] along with structural characterization data by mass spectrometry. Provision of the peptide nucleic acid derivative of the present invention in Table 1 is to exemplify the PNA derivative of Formula I, and should not be interpreted to limit the scope of the present invention.

[0160] “ASO 1” has a 14-mer complementary overlap with the 14-mer sequence marked “bold” and “underlined” within the 30-mer RNA sequence of [(5′.fwdarw.3′) UCCAAGCCAUAUAUG|gugaguauggggaaa] spanning the junction of “exon 5” and “intron 5” in the human MMP-1 pre-mRNA. Thus “ASO 1” possesses a 7-mer overlap with “exon 5” and a 7-mer overlap with “intron 5” within the human MMP-1 pre-mRNA.

[0161] Binding Affinity of “ASO 1” for Complementary DNA

[0162] T.sub.m values were determined on a UV/Vis spectrometer as follows. A mixed solution of 4 μM PNA oligomer and 4 μM complementary 14-mer DNA in 4 mL aqueous buffer (pH 7.16, 10 mM sodium phosphate, 100 mM NaCl) in 15 mL polypropylene falcon tube was incubated at 90° C. for a minute and slowly cooled down to ambient temperature. Then the solution was transferred into a 3 mL quartz UV cuvette equipped with an air-tight cap, and subjected to a T.sub.m measurement at 260 nm on a UV/Visible spectrophotometer (Agilent 8453). The absorbance changes at 260 nm were recorded with increasing the temperature of cuvette by either 0.5 or 1.0° C. per minute. From the absorbance vs temperature curve, the temperature showing the largest increase rate in absorbance was read out as the melting temperature T.sub.m between PNA and DNA. The 14-mer complementary DNAs for T.sub.m measurement were purchased from Bioneer (www.bioneer.com, Dajeon, Republic of Korea) and used without further purification.

[0163] “ASO 1” showed a T.sub.m value of 72.67° C. for the duplex with the 14-mer complementary DNA

Examples for Biological Activities of PNA Derivatives of Formula I

[0164] PNA derivatives in this invention were evaluated for in vitro MMP-1 antisense activities in human dermal fibroblasts (HDF) by use of real-time quantitative polymerase chain reaction (RT qPCR) and so on. The biological examples were provided as examples to illustrate the biological profiles of the PNA derivatives of Formula I, and therefore should not be interpreted to limit the scope of the current invention.

Example 1. Inhibition of MMP-1 mRNA Formation by “ASO 1” in HDF

[0165] “ASO 1” was evaluated by Western blotting for its ability to down-regulate the MMP-1 mRNA formation in HDF as described below.

[0166] [Cell Culture & ASO Treatment] HDF cells were maintained in Fibroblast Basal Medium (ATCC PCS-201-030) supplemented with fibroblast growth kit-low serum (ATCC PCS-201-041) and 1% streptomycin/penicillin, which was grown at 37° C. and under 5% CO2 condition. HDF cells (3×10.sup.5) stabilized for 24 hours in 60 mm culture dish were incubated for 24 hours with “ASO 1” at 0 (negative control) and 100 aM to 1 μM.

[0167] [RNA Extraction & cDNA Synthesis] Total RNA was extracted using 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 of RNA by use of PrimeScript™ 1.sup.st strand cDNA Synthesis Kit (Takara, Cat. No. 6110A). To a mixture of 400 ng of RNA, 1 μl of random hexamer, and 1 μl of dNTP (10 mM) in PCR tube was added DEPC-treated water 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, successively.

[0168] [Quantitative Real-Time PCR] In order to evaluate the expression level of human MMP-1 mRNA real-time qPCR was performed with synthesized cDNA by use of Taqman probe. The mixture of cDNA, Taqman probe, IQ supermix (BioRad, Cat. No. 170-8862), and nuclease free water in PCR tube was under reaction by use of CFX96 Touch Real-Time system (BioRad) according to the cycle conditions specified as follows: 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 every cycle and the result of PCR was evaluated by the melting curve. After the threshold cycle (Ct) of each gene was standardized by that of GAPDH, the change of Ct was compared and analyzed.

[0169] [MMP-1 mRNA Decrease by ASO] As can be seen in FIG. 8, compared to control experiment the amount of MMP-1 mRNA reduced were 65% at 1 μM treatment of “ASO 1” and 10 to 15% at 100 aM to 10 nM μM treatment of “ASO 1”, respectively.

Example 2. Inhibition of MMP-1 Protein Expression by “ASO 1” in HDF

[0170] “ASO 1” was evaluated by Western blotting for its ability to down-regulate the MMP-1 protein expression in HDF as described below.

[0171] [Western Blotting] HDF cells were grown as Example 1 and 48 hours later cells were 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. The protein was quantified with BCA solution (Thermo, Cat. No. 23225) and purified by 8% SDS-PAGE Gel. The protein was transferred on PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was probed with an anti-MMP-1 (SantaCruz, Cat. No. 58377) and anti-β-actin (Sigma, Cat. No. A3854) as a primary antibody, and goat anti-mouse (CST, Cat. No. 7076V) was used as a secondary antibody. SuperSignal™ West Pico (PierAce, USA) was utilized for the detection of chemiluminescent signal and the signal intensity was measured by using Gel Doc system (ATTO). Based on Western blotting results of each bands, the relative expression levels of MMP-1 were quantified with Image-J program.

[0172] [Inhibition of MMP-1 Protein Expression by ASO] As shown in FIGS. 9a and 9b, compared to control experiment the amount of MMP-1 protein expression level reduced was 20 to 50% at 100 aM to 1 μM treatment of “ASO 1” Therefore, “ASO 1” was proved to show its ability to down-regulate the MMP-1 protein expression in HDF.

Example 3. Enhancement of Collagen Protein Expression by “ASO 1” in HDF

[0173] “ASO 1” was evaluated by Western blotting for its ability to up-regulate the type I collagen protein expression in HDF associated with MMP-1 protein expression reduction as described below.

[0174] [Western Blotting] HDF cells were grown as Example 1 and 48 hours later cells were 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. The protein was quantified with BCA solution (Thermo, Cat. No. 23225) and purified by 8% SDS-PAGE Gel. The protein was transferred on PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was probed with an anti-MMP-1 (SantaCruz, Cat. No. 58377) and anti-β-actin (Sigma, Cat. No. A3854) as a primary antibody, and rabbit anti-goat (Santacruz, Cat. No. 2768) was used as a secondary antibody. SuperSignal™ West Pico (PierAce, USA) was utilized for the detection of chemiluminescent signal and the signal intensity was measured by using Gel Doc system (ATTO). Based on Western blotting results of each bands, the relative expression levels of MMP-1 were quantified with Image-J program.

[0175] [Enhancement of Type I Collagen Protein Expression by ASO] As shown in FIGS. 10a and 10b, compared to control experiment the amount of type I collagen protein expression level enhanced was more than 30% at 100 aM to 1 μM treatment of “ASO 1” Therefore, WIMP-1 protein expression reduction induced by “ASO 1” was proved to show its ability to up-regulate the type I collagen protein expression in HDF.

Example 4. Inhibition of MMP-1 Protein Expression by “ASO 1” in Extracellular Fluid (Western Blotting)

[0176] MMP-1 protein expression reduction induced by “ASO 1” in cell, as a result, may affect WIMP-1 protein expression level secreted outside cell. In that sense, “ASO 1” was evaluated by Western blotting for its ability to down-regulate the MMP-1 protein expression in culture fluid of cells at 48 hours after treating “ASO 1” as described below.

[0177] [Western Blotting] HDF cells were grown as Example 1 and 48 hours later collected culture fluid of cells was purified by 8% SDS-PAGE Gel. The separated protein was transferred on PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was probed with an anti-MMP-1 (SantaCruz, Cat. No. 58377) as a primary antibody and goat anti-mouse (CST, Cat. No. 7076V) was used as a secondary antibody. SuperSignal™ West Pico (PierAce, USA) was utilized for the detection of chemiluminescent signal and the signal intensity was measured by using Gel Doc system (ATTO). Based on Western blotting results of each bands, the relative expression levels of MMP-1 were quantified with Image-J program.

[0178] [Inhibition of MMP-1 Protein Expression by ASO] As shown in FIGS. 11a and 11b, compared to control experiment the amount of MMP-1 protein expression level reduced was 10 to 60% at 100 μM to 1 μM treatment of “ASO 1” in extracellular fluid. Therefore, “ASO 1” was proved to show its ability to down-regulate the MMP-1 protein expression in extracellular fluid.

Example 5. Enhancement of Collagen Protein Expression by “ASO 1” in Extracellular Fluid

[0179] “ASO 1” was evaluated by Western blotting for its ability to up-regulate the type I collagen protein expression in extracellular fluid as described below.

[0180] [Western Blotting] HDF cells were grown as Example 1 and 48 hours later collected culture fluid of cells was purified by 8% SDS-PAGE Gel. The separated protein was transferred on PVDF membrane (polyvinylidene fluoride membrane) (Millipore, Cat. No. IPVH00010), which was blocked in skim milk buffer for 1 hour. The membrane was probed with an anti-MMP-1 (SantaCruz, Cat. No. 58377) as a primary antibody and rabbit anti-goat (Santacruz, Cat. No. 2768) was used as a secondary antibody. SuperSignal™ West Pico (PierAce, USA) was utilized for the detection of chemiluminescent signal and the signal Intensity was measured by using Gel Doc system (ATTO). Based on Western blotting results of each bands, the relative expression levels of MMP-1 were quantified with Image-J program.

[0181] [Enhancement of Type I Collagen Protein Expression by ASO] As shown in FIGS. 12a and 12b, compared to control experiment the amount of type I collagen protein expression level enhanced was 20% at 1 pM and 80% at 1 μM treatment of “ASO 1”, respectively. Therefore, MMP-1 protein expression reduction induced by “ASO 1” in HDF was proved to show its ability to up-regulate the type I collagen protein expression in extracellular fluid.

Example 6. Inhibition of MMP-1 Protein Expression by “ASO 1” in Extracellular Fluid (ELISA)

[0182] MMP-1 protein expression reduction induced by “ASO 1” in cell, as a result, may affect MMP-1 protein expression level secreted outside cell. In that sense, “ASO 1” was evaluated by enzyme linked immunosorbent assay (ELISA) for its ability to down-regulate the MMP-1 protein expression in culture fluid of cells at 48 hours after treating “ASO 1” as described below.

[0183] [ELISA] HDF cells were grown as Example 1 and 48 hours later in collected culture fluid of cells MMP-1 expression level was evaluated through absorbance (Sunrise, TECAN) with human MMP-1 ELISA kit (abcam, Cat. No. ab100603) according to the manufacturer's instruction.

[0184] [Inhibition of MMP-1 Protein Expression by ASO] As shown in FIG. 13, compared to control experiment the amount of MMP-1 protein expression level reduced was 15 to 30% at 100 pM to 10 nM and 50% at 1 μM treatment of “ASO 1”, respectively. Therefore, “ASO 1” was proved to show its ability to down-regulate the MMP-1 protein expression in extracellular fluid.

Example 7. Preparation of Topical Serum Containing Compound of Formula I

[0185] (w/w %)

[0186] A compound of Formula I, for example “ASO 1” was formulated as a serum for topical application to subjects. The topical serum was prepared as described below. Given that there are lots of variations of topical serum possible, this preparation should be taken as an example and should not be interpreted to limit the scope of the current invention.

TABLE-US-00002 Amount Part No. Substance Name (w/w %) A 1 PEG-40 Hydrogenated Castor Oil 0.500 2 Ethylhexylglycerin 0.200 3 Perfume 0.050 4 Glycerin 5.000 B 5 Butylene Glycol 7.000 6 Dipropylene Glycol 2.000 7 1,2-Hexandiol 0.200 8 Arginine 0.150 9 Deionized Water 58.615 C 10 Sodium Hyaluronate 0.100 11 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.100 12 Carbomer 0.060 13 Ammonium Acryloyldimethyltaurate/VP 0.025 Copolymer 14 Deionized Water 23.000 D 15 β-glucan 2.000 16 Biosaccharide Gum-1 0.500 17 “ASO 1” 1 pM + Polysorbate 80 0.1% 0.500 SUM 100.000

[0187] In a separate beaker, the mixed substances of part A and part B at 25° C., respectively, were dissolved. Part A and part B was mixed and emulsified by use of 3,600 rpm homogenizer at 25° C. for 5 minutes. Emulsified part C was filtered through 50 mesh and the filtrate was added to the mixture of part A and B. The resulting mixture was emulsified by use of 3,600 rpm homogenizer at 80° C. for 5 minutes. After addition of part D to the mixture of part A, B, and C, the resulting mixture was emulsified by use of 2,500 rpm homogenizer at 25° C. for 3 minutes. Finally make sure homogeneous dispersion and complete defoamation.

Example 8. Preparation of Topical Cream Containing Compound of Formula I

[0188] (w/w %)

[0189] A compound of Formula I, for example “ASO 1” was formulated as a cream for topical application to subjects. The topical cream was prepared as described below. Given that there are lots of variations of topical cream possible, this preparation should be taken as an example and should not be interpreted to limit the scope of the current invention.

[0190] In a separate beaker, were dissolved substances of part A at 80° C. and part B at 85° C., respectively. Part A and part B was mixed and emulsified by use of 3,600 rpm homogenizer at 80° C. for 5 minutes. After addition of part C and D to the mixture of part A and B, the resulting mixture was emulsified by use of 3,600 rpm homogenizer at 80° C. for 5 minutes. After addition of part E to the mixture of part A, B, C, and D at 35° C., the resulting mixture was emulsified by use of 3,600 rpm homogenizer at 35° C. for 3 minutes. Finally make sure homogeneous dispersion and complete defoamation at 25° C.

TABLE-US-00003 Amount Part No. Substance Name (w/w %) A 1 Shea Butter 15.000 2 Simmondsia Chinensis (Jojoba) Seed Oil 8.000 3 Caprylic/Capric Triglyceride 6.000 4 Sunflower Seed Oil 4.000 5 Cetearyl Alcohol 3.000 6 Glyceryl Stearate 2.500 7 PEG-100 Stearate 2.500 8 Macadamia Seed Oil 2.000 9 Polysorbate 80 0.500 B 10 1,3-Propanediol 2.000 11 Glycerin 1.000 12 Deionized Water 51.630 C 13 Corn Starch 0.300 D 14 Hydroxyethyl Acrylate/Sodium 0.300 Acryloyldimethyl Tau E 15 1,2-Hexanediol 0.300 16 Ethylhexylglycerin 0.300 17 Tocopheryl Acetate 0.100 18 Perfume 0.070 19 “ASO 1” 1 pM + Polysorbate 80 0.1% 0.500 SUM 100.000