Acetyl-CoA carbosylase2 antisense oligonucleotides

Abstract

The present invention provides the peptide nucleic acid derivative which targets 5′ splice site of the human ACC2 pre-mRNA “exon 12”. The peptide nucleic acid derivatives in the present invention strongly induce splice variants of the human ACC2 mRNA in cell and are very useful to treat conditions or disorders of skin aging associated with the human ACC2 protein.

Claims

1. A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof, for inducing exon skipping within human ACC2 pre-mRNA: ##STR00009## wherein, n is an integer between 11 and 15; the compound of Formula I possesses at least a 10-mer complementary overlap with the 18-mer pre-mRNA sequence of [(5′-3′) GGCCAUUUCGUCAGUAUC] (SEQ ID NO: 7) in the human ACC2 pre-mRNA; the compound of Formula I is fully complementary to the human ACC2 pre-mRNA, or partially complementary to the human ACC2 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 are hydrido radical; X and Y independently represent hydrido, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; and 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 represented by Formula II, Formula III, or Formula IV ##STR00010## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are hydrido radical; L1 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—; and, L2 and L3 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)r, and —(CH.sub.2).sub.8—.

2. The peptide nucleic acid derivative according to claim 1, which is selected from the group of peptide nucleic acid derivatives provided below, or a pharmaceutically acceptable salt thereof: (N.fwdarw.C) Fethoc-CTG(6)-ACG(6)-AA(5)A-TG(6)G-C(1O2)C—NH.sub.2 [SEQ ID NO: 1]; (N.fwdarw.C) Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-N H.sub.2 [SEQ ID NO: 5]; and (N.fwdarw.C) Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-C(1O2)C—NH.sub.2 [SEQ ID NO: 6] wherein, A, G, T, and C are monomers of peptide nucleic acid with a natural nucleobase of adenine, thymine, guanine and cytosine, respectively; C(1O2), A(5), and G(6) are monomers of peptide nucleic acid with an unnatural nucleobase as follows: ##STR00011## and “Fethoc-” is the abbreviation for “[2-(9-fluorenyl)ethyl-1-oxy]carbonyl”.

3. A method of inducing skipping of exon 12 in the human ACC2 pre-mRNA to yield a ACC2 mRNA splice variant lacking ACC2 exon 12 in cells in vitro, comprising contacting the cells with the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

4. A cosmetic composition for improving conditions or disorders associated directly with human ACC2 gene transcription through downregulation of the ACC2 gene transcription, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

5. A cosmetic composition for improving skin aging by down-regulating human ACC2 gene expression, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

Description

BRIEF EXPLANATION OF DRAWINGS

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

(2) FIGS. 2a-2e. Examples of substituents selectable for the peptide nucleic acid derivative of Formula I.

(3) FIG. 3. Chemical structures of PNA monomers with natural or modified nucleobase.

(4) FIG. 4. Chemical structure of “ASO 1”.

(5) FIG. 5. Chemical structures of Fmoc-PNA monomers used to synthesize the PNA derivatives of this invention.

(6) FIGS. 6a-6b. Cis-reverse phase HPLC chromatograms of “ASO 1” before and after HPLC purification, respectively.

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

(8) FIG. 8. Exon Skipping of ACC2 mRNA by “ASO 1” in C2C12.

(9) FIG. 9. Inhibition of ACC2 mRNA Levels by “ASO 1” in C2C12.

(10) FIG. 10. Inhibition of ACC2 mRNA Levels by “ASO 6” in C2C12.

(11) FIG. 11. Inhibition of ACC2 mRNA Levels by “ASO 5” in C2C12.

(12) FIG. 12. Production of malonyl-CoA through Acetyl-CoA carboxylase (ACC) catalyzing the carboxylation of acetyl-CoA.

(13) FIG. 13. An exemplified numbered exons and introns.

(14) FIG. 14. A schematic summary of Pre-mRNA processed into mRNA by “splicing”.

(15) FIG. 15. A scheme illustrating 3′ splice site and 5′ splice site.

(16) FIG. 16. Brief Illustration of the Scheme 1.

BEST MODE FOR CARRYING OUT THE INVENTION

(17) General Procedures for Preparation of PNA Oligomers

(18) 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. Fmoc is {(9-fluorenyl)methyloxy}carbonyl. The solid support employed in this study was H-Rink Amide-ChemMatrix purchased from PCAS BioMatrix Inc. (Quebec, Canada). Fmoc-PNA monomers with a modified nucleobase were synthesized as described in the prior art [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 Cis-reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

(19) 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 in FIG. 16.

(20) [Activation of H-Rink-ChemMatrix Resin] When the amine on the resin was not protected with Fmoc, 0.01 mmol (ca 20 mg resin) of the ChemMatrix resin in 1.5 mL 20% piperidine/DMF was vortexed in a libra tube for 20 min, and the DeFmoc solution was filtered off. The resin was washed for 30 sec each in series with 1.5 mL methylene chloride (MC), 1.5 mL dimethylformamide (DMF), 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The resulting free amines on the solid support were subjected to coupling with an Fmoc-PNA monomer.

(21) [DeFmoc] When the amine on the resin was protected with Fmoc, the suspension of 0.01 mmol (ca 20 mg) of the resin in 1.5 mL 20% piperidine/DMF was vortexed 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.

(22) [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 0.1 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. 5. The Fmoc-PNA monomers with a modified nucleobase are provided in FIG. 5 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.

(23) [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.

(24) [Introduction of “Fethoc-” Radical in N-Terminus] “Fethoc-” radical was introduced to the N-terminus by reacting the free amine on the resin with “Fethoc-OSu” by the following method. The suspension of the resin in the solution of 0.1 mmol of Fethoc-OSu and 0.1 mmol DIEA in 1 mL anhydrous MDF was vortexed for 1 hr, and the solution was filtered off. 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 structure of “Fethoc-OSu” [CAS No. 179337-69-0, C.sub.20H.sub.17NO.sub.5, MW 351.36] used in the present invention is provided as follows.

(25) ##STR00008##

(26) [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.

(27) [HPLC Analysis and Purification] Following a cleavage from resin, the crude product of a PNA derivative was purified by Cis-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.

(28) Synthetic Examples for PNA Derivative of Formula I

(29) In order to complementarily target the 5′ splice site of “exon 12” in the human ACC2 pre-mRNA, PNA derivatives of this invention were prepared according to the synthetic procedures provided above or with minor modifications. Provision of such PNA derivatives targeting the human ACC2 pre-mRNA is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

(30) TABLE-US-00002 TABLE 1 PNA derivatives complementarily targeting the 5′ splice site of “exon 12” in the human ACC2 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 Fethoc-CTG(6)—ACG(6)—AA(5)A—TG(6)G—C(1O2)C—NH.sub.2 4549.07 4549.08 1 ASO Fethoc-TA(5)C(1O2)—TGA(5)—CGA(5)—AA(5)T—G(6)GC(1O2)— 5289.43 5289.38 2 C—NH.sub.2 ASO Fethoc-TA(5)C—TG(5)A-C(1O2)GA(5)—AA(5)T—G(5)G—NH.sub.2 4661.14 4661.18 3 ASO Fethoc-AC(1O2)T—GA(5)C-GA(5)A—A(5)TG(5)—GC(1O2)—NH.sub.2 4658.11 4658.10 4 ASO Fethoc-CTG(6)—AC(1O2)G—A(5)AA(5)—TG(6)G—NH.sub.2 4047.86 4047.87 5 ASO Fethoc-CTG(6)—AC(1O2)G—A(5)AA(5)—TG(6)G—C(1O2)C—NH.sub.2 4647.12 4647.12 6 .sup.atheoretical exact mass, .sup.bobserved exact mass

(31) Table 1 provides PNA derivatives complementarily targeting the 5′ splice site of “exon 12” in the human ACC2 pre-mRNA read out from the human ACC2 gene [NCBI Reference Sequence: NG_046907] along with structural characterization data by mass spectrometry. Provision of the peptide nucleic acid derivatives of the present invention in Table 1 is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

(32) “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′) GGAAGAGGCCAUUUC|gucaguaucuccuuc] (SEQ. NO. 8) spanning the junction of “exon 12” and “intron 12” in the human ACC2 pre-mRNA. Thus “ASO 1” possesses a 9-mer overlap with “exon 12” and a 5-mer overlap with “intron 12” within the human ACC2 pre-mRNA.

(33) Binding Affinity of “ASO” for Complementary DNA

(34) The PNA derivatives of Formula I were evaluated for their binding affinity for 10-mer DNAs complementarily targeting either the N-terminal or C-terminal. The binding affinity was assessed by T.sub.m value for the duplex between PNA and 10-mer complementary DNA. The duplex between PNA derivatives and fully complementary DNAs show T.sub.m values too high to be reliably determined in aqueous buffer solution, since the buffer solution tends to boil during the T.sub.m measurement. T.sub.m values for full length PNAs can be predicted and compared based on the T.sub.m value for the duplex between PNA and 10-mer complementary DNA.

(35) T.sub.m values were determined on a UV/Vis spectrometer as follows. A mixed solution of 4 M PNA oligomer and 4 μM complementary 10-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 few 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 the cuvette was mounted on an Agilent 8453 UV/Visible spectrophotometer. The absorbance changes at 260 nm were recorded with increasing the temperature of the cuvette by either 0.5 or 1° C. per minute. From the absorbance vs temperature curve, the temperature showing the largest increase rate in absorbance was read out as the T.sub.m between PNA and 10-mer DNA. The DNAs for T.sub.m measurement were purchased from Bioneer (www.bioneer.com, Dajeon, Republic of Korea) and used without further purification.

(36) Observed T.sub.m values of the PNA derivatives of Formula I are very high for a complementary binding to 10-mer DNA as provided in Table 2.

(37) TABLE-US-00003 TABLE 2 T.sub.m values between PNAs and 10-mer complementary DNA targeting either the N-terminal or the C-terminal of PNA. T.sub.m Value, ° C. 10-mer DNA 10-mer DNA PNA against N-Terminal against C-Terminal ASO 1 72.80 79.60 ASO 2 82.99 82.01 ASO 3 76.03 78.99 ASO 4 80.01 82.01

(38) For example, “ASO 1” showed a T.sub.m value of 72.80° C. for the duplex with the 10-mer complementary DNA targeting the N-terminal 10-mer in the PNA as marked “bold” and “underlined” in [(N.fwdarw.C) Fethoc-CTG(6)-ACG(6)-AA(5)A-TG(6)G-C(1O2)C—NH.sub.2] (SEQ. NO. 1). In the meantime, “ASO 1” showed a T.sub.m of 79.60° C. for the duplex with the 10-mer complementary DNA targeting the C-terminal 10-mer in the PNA as marked “bold” and “underlined” in [(N.fwdarw.C) Fethoc-CTG(6)-ACG(6)-AA(5)A-TG(6)G-C(1O2)C—NH.sub.2] (SEQ. NO. 1).

(39) Examples for Biological Activities of PNA Derivatives of Formula I

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

Example 1. Exon Skipping Induced by “ASO 1” in C2C12

(41) “ASO 1” was evaluated for its ability to induce the skipping of ACC2 “exon 12” in C2C12 cells as described below.

(42) [Cell Culture & ASO Treatment] C2C12 cells (2×10.sup.5) (Cat. No. CRL-1772, ATCC) were grown in 60 mm culture dish containing DMEM medium (Dulbecco Modified Eagle Medium: DMEM) (Cat. No. 12-604F, Lonza) supplemented with 10% FBS (Fetal Bovine Serum) (Cat. No. 10099-41, GIBCO) and 1% streptomycin/penicillin (Cat. No. 15140-122, GIBCO) under 5% CO.sub.2 atmosphere at 37° C. The cells were treated either with nothing (negative control) or with an aliquot of aqueous stock solution of “ASO 1” for 5 hours at 100 zM to 1 fM.

(43) [RNA Extraction & Nested PCR] 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 200 ng of RNA by use of SuperScript™ III One-Step RT-PCR System (Cat. No. 12574-018, Invitrogen). To a mixture of 200 ng of RNA, 25 microliter of 2× Reaction Mix buffer, 2 microliter of SuperScript III™ RT/Platinum Taq Mix, 1 microliter of 10 M (micromole conc.) Exon 9 Forward Primer (5′-TTTTCCGACAAGTGCAGAG-3′) (SEQ. NO. 9), and 1 microliter of 10 Mm Exon 15 Reverse Primer (5′-AACGTCCACAATGTTCAG-3′) (SEQ. NO. 10) in PCR tube was added autoclaved distilled water to a total volume of 50 microliter. After reaction at 60° C. for 30 minutes and at 94° C. for 2 minutes, 30 cycles PCR process at 94° C. for 15 seconds, at 50° C. for 30 seconds, and at 68° C. for 1 minute afforded the first crude product. The mixture of 1 microliter of the crude product, 1 microliter of 10 μM Exon 10 Forward Primer (5′-GAG TAC TTA TAC AGC CAG G-3′) (SEQ. NO. 11), and 1 microliter of 10 μM Exon 14 Reverse Primer (5′-TTC TGA ACA TCG CGT CTG-3′) (SEQ. NO. 12) was reacted, using PyroHostStart Taq Polymerase Kit (Cat. No. K-2611-FCG) according to the manufacturer's instructions, at 95° C. for 2 minutes, and then was under PCR process at 95° C. for 30 seconds, at 47° C. for 1 minute, and at 72° C. for 20 seconds.

(44) [Identification of Exon Skipping Products Electrophoresis] The PCR products (10 microliter) were subjected to electrophoretic separation on a 2% agarose gel. The target bands from “ASO 1” treatment were collected and analyzed by Sanger Sequencing to evaluate exon skipping sequence.

(45) [Exon Skipping Induced by “ASO 1” ] As can be seen in FIG. 8, the cells treated with “ASO 1” at 0.1 aM to 1 fM concentration-dependently yielded the splice variant ACC2 mRNA lacking exon 11.

Example 2. Inhibition of ACC2 mRNA Formation by “ASO 1” in C2C12

(46) “ASO 1” was evaluated by Real-Time qPCR for its ability to down-regulate the ACC2 mRNA formation in C2C12 as described below.

(47) [Cell Culture & ASO Treatment] C2C12 cells (Cat. No. CRL-1772, ATCC) were maintained in Dulbecco Modified Eagle Medium (DMEM, Cat. No. 12-604F, Lonza) supplemented with 10% Fetal Bovine Serum (Cat. No. 10099-41, GIBCO) and 1% streptomycin/penicillin (Cat. No. 15140-122, GIBCO), which was grown at 37° C. and under 5% CO.sub.2 condition. C2C12 cells (2×10.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 zM to 1 fM.

(48) [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™ 1st strand cDNA Synthesis Kit (Takara, Cat. No. 6110A). To a mixture of 400 ng of RNA, 1 microliter of random hexamer, and 1 microliter of dNTP (10 mM) in PCR tube was added DEPC-treated water to a total volume of 10 microliter, which was reacted at 65° C. for 5 minutes. cDNA was synthesized by adding 10 microliter of PrimeScript RTase to the reaction mixture and reacting at 30° C. for 10 minutes and at 42° C. for 60 minutes, successively.

(49) [Quantitative Real-Time PCR] In order to evaluate the expression level of human ACC2 mRNA real-time qPCR was performed with synthesized cDNA by use of Tagman probe. The mixture of cDNA, Taqman probe (Thermo, Mm01204651), 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: at 95° C. for 3 min (primary denaturation) followed by 50 cycles of 10 sec at 95° C. (denaturation) and 30 sec at 60° C. (annealing and polymerization). Fluorescence intensity was measured at the end of every cycle and the result of PCR was evaluated by the melting curve. After the threshold cycle (Ct) of each gene was standardized by that of GAPDH, the change of Ct was compared and analyzed.

(50) [ACC2 mRNA Decrease by “ASO 1” ] As can be seen in FIG. 9, compared to control experiment the amount of ACC2 mRNA was reduced at 100 zM to 1 fM treatment of “ASO 1”, concentration-dependently, and statistically significant 30% of reduction was observed at 1 fM treatment of “ASO 1”. (Student T-test was done to check the statistical significance of the findings)

Example 3. Inhibition of ACC2 mRNA Formation by “ASO 6” in C2C12

(51) “ASO 6” was evaluated by Real-Time qPCR for its ability to down-regulate the ACC2 mRNA formation in C2C12 as described below.

(52) [Cell Culture & ASO Treatment] C2C12 cells (Cat. No. CRL-1772, ATCC) were maintained in Dulbecco Modified Eagle Medium (DMEM, Cat. No. 12-604F, Lonza) supplemented with 10% Fetal Bovine Serum (Cat. No. 10099-41, GIBCO) and 1% streptomycin/penicillin (Cat. No. 15140-122, GIBCO), which was grown at 37° C. and under 5% CO.sub.2 condition. C2C12 cells (2×10.sup.5) stabilized for 24 hours in 60 mm culture dish were incubated for 24 hours with “ASO 6” at 0 (negative control) and 100 zM to 1 fM.

(53) [RNA Extraction & cDNA Synthesis] Total RNA was extracted using RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions from “ASO 6” treated cells and cDNA was prepared from 400 ng of RNA by use of PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Cat. No. 6110A). To a mixture of 400 ng of RNA, 1 microliter of random hexamer, and 1 microliter of dNTP (10 mM) in PCR tube was added DEPC-treated water to a total volume of 10 microliter, which was reacted at 65° C. for 5 minutes. cDNA was synthesized by adding 10 microliter of PrimeScript RTase to the reaction mixture and reacting at 30° C. for 10 minutes and at 42° C. for 60 minutes, successively.

(54) [Quantitative Real-Time PCR] In order to evaluate the expression level of human ACC2 mRNA real-time qPCR was performed with synthesized cDNA by use of Tagman probe. The mixture of cDNA, Taqman probe (Thermo, Mm01204651), 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: at 95° C. for 3 min (primary denaturation) followed by 50 cycles of 10 sec at 95° C. (denaturation) and 30 sec at 60° C. (annealing and polymerization). Fluorescence intensity was measured at the end of every cycle and the result of PCR was evaluated by the melting curve. After the threshold cycle (Ct) of each gene was standardized by that of GAPDH, the change of Ct was compared and analyzed.

(55) [ACC2 mRNA Decrease by “ASO 6” ] As can be seen in FIG. 10, the amount of ACC2 mRNA was reduced at 100 zM to 1 fM treatment of “ASO 6”, concentration-dependently. Compared to the control experiment, statistically significant 30% and 50% reduction was observed at 1 aM and 1 fM treatment of “ASO 6”, respectively. (Student T-test was done to check the statistical significance of the findings)

Example 4. Inhibition of ACC2 mRNA Formation by “ASO 5” in C2C12

(56) “ASO 5” was evaluated by the same method as described below.

(57) [Cell Culture & ASO Treatment] C2C12 cells (Cat. No. CRL-1772, ATCC) were maintained in Dulbecco Modified Eagle Medium (DMEM, Cat. No. 12-604F, Lonza) supplemented with 10% Fetal Bovine Serum (Cat. No. 10099-41, GIBCO) and 1% streptomycin/penicillin (Cat. No. 15140-122, GIBCO), which was grown at 37° C. and under 5% CO.sub.2 condition. C2C12 cells (2×10.sup.5) stabilized for 24 hours in 60 mm culture dish were incubated for 24 hours with “ASO 5” at 0 (negative control) and 100 zM to 1 fM.

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

(59) [Quantitative Real-Time PCR] In order to evaluate the expression level of human ACC2 mRNA real-time qPCR was performed with synthesized cDNA by use of Tagman probe. The mixture of cDNA, Taqman probe (Thermo, Mm01204651), 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: at 95° C. for 3 min (primary denaturation) followed by 50 cycles of 10 sec at 95° C. (denaturation) and 30 sec at 60° C. (annealing and polymerization). Fluorescence intensity was measured at the end of every cycle and the result of PCR was evaluated by the melting curve. After the threshold cycle (Ct) of each gene was standardized by that of GAPDH, the change of Ct was compared and analyzed.

(60) [ACC2 mRNA Decrease by “ASO 5” ] As can be seen in FIG. 11, the amount of ACC2 mRNA was reduced at 100 zM to 1 fM treatment of “ASO 5”, concentration-dependently. Compared to the control experiment, statistically significant 30% and 42% reduction was observed at 1 aM and 1 fM treatment of “ASO 5”, respectively. (Student T-test was done to check the statistical significance of the findings)

Example 5. Preparation of Body Lotion Containing Compound of Formula I. (w/w %)

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

(62) In a separate beaker, mixed substances of part A and part B were dissolved at 80° C., respectively. Part A and part B was mixed and emulsified by use of 3,600 rpm homogenizer at 80° 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 at 35° 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.

(63) TABLE-US-00004 TABLE 3 Example of Composition for Body Lotion Containing Compound of Formula I. (w/w %) Part No. Substance Name Amount A 1 Polyglyceryl-3 Methylglucose Distearate 0.700 2 Glyceryl Stearate 0.300 3 Cetearyl Alcohol 1.000 4 Shea Butter 1.000 5 Caprylic/Capric Triglyceride 3.000 6 Dicaprylyl Carbonate 4.000 7 Dimethicone 0.500 8 Ethylhexylglycerin 0.300 B 9 Glycerin 5.000 10 Propanediol 5.000 11 1,2-Hexanediol 0.300 12 Arginine 0.160 13 Deionized Water 62.110 C 14 Sodium Acrylate/Sodium Acryloyldimethyl 0.300 Tau Copolymer 15 Carbomer 0.200 16 Xanthan Gum 0.030 17 Deionized Water 13.000 D 18 Perfume 0.100 19 Oligomer [1000 fM] + POLYSORBATE 80 [0.1%] 3.000 SUM 100.000

Example 6. Preparation of Face Cream Containing Compound of Formula I. (w/w %)

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

(65) TABLE-US-00005 TABLE 4 Example of Composition for Face Cream Containing Compound of Formula I. (w/w %) Part No. Substance Name Amount (%/%) A 1 Caprylic/Capric Triglyceride 2.000 2 Glyceryl Stearate/Polyglyceryl-10 Stearate 10.000 3 Cetearyl Alcohol 2.000 4 Ethylhexylglycerin 0.300 B 10 Glycerin 5.000 11 1,2-Hexanediol 0.300 12 Deionized Water 78.900 C 14 Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Tau Copolymer 1.000 D 19 Oligomer [1000 fM] + POLYSORBATE 80 [0.1%] 0.500 SUM 100.000
In a separate beaker, mixed substances of part A and part B were dissolved at 80° 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 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 at 35° C., the resulting mixture was emulsified by use of 3,600 rpm homogenizer at 35° C. for 5 minutes. Finally make sure homogeneous dispersion and complete defoamation at 25° C.