Quantitative Real-time PCR
mRNA was extracted from the human skin model tissues (Maxwell® RSC simplyRNS Tissue Kit; Promega) followed by cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit; ThermoFisher Scientific) and quantitative real-time PCR (TaqMan Fast Advanced Master Mix, ThermoFisher Scientific). Gene expression analyses were performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) with real-time PCR system QuantStudio7 Flex (ThermoFisher Scientific). The assay mix used for studies were: GAPDH (Cat#: Hs02758991_ g1), COL1A1 (Cat#: Hs001640004_m1); COL3A1 (Cat#: Hs00943809_m1), COL6A1 (Cat#: Hs01095585_m1), COL7A1 (Cat#: Hs00164310_m1), TGFB1 (Cat#: Hs00998133_m1), DCN (Cat#: Hs00754870_s1), ELN (Cat#: Hs00355783_m1), FBN1 (Cat# Hs00171191_m1), FBLN5 (Cat#: Hs00197064_m1 ), MFAP1 (Cat#: Hs00195537_m1), LOXL1 (Cat# Hs00935937_m1), POMP (Cat#: Hs01106088_m1), PSMB5 (Cat#: Hs00605652_m1), PSMB6 (Cat#: Hs00382586_m1), ATG5 (Cat#: Hs00169468_m1), ATG7 (Cat#: Hs00893766_m1), ATG12 (Cat#: Hs04980076_s1), BECN1 (Cat#: Hs01007018_m1), HSPA8(Cat#: Hs03044880_gH).
Elastase Assay
The assay employed was based on methods previously described in literature.8 In brief, human skin model tissue was homogenized and solubilized in 0.1% Triton-X 100, 0.2 M Tris– HCl (pH 8.0) buffer, followed by ultrasonication and subsequent centrifugation at 2000 x g for 10 minutes to obtain supernatants for enzyme assay. To measure elastase activity, 2 μL of substrate (62.5mM, N-succinyl-tri-alanyl-p nitroanilide from Sigma-Aldrich) was incubated with 100 μL lysate solution at 37°C for 2 hours. The amount of released nitroaniline was measured by determining absorbance at 410 nm using a spectrophotometer (Molecular Devices).
Clinical Study Design
Study Design
A randomized, double-blind, regimen-controlled study was conducted to assess the efficacy and tolerability of NCC as a topical treatment for the neck and décolletage. Criteria for study participation included female subjects aged 45–70 years, presenting moderate to severe overall skin texture on the neck and moderate to severe skin tone unevenness on the décolletage.
Institutional Review Board approval (IntegReview IRB, Austin, TX) was obtained prior to conduct of any study procedures.The conduct of the study followed all applicable guidelines for the protection of human subjects for research as per 21 CFR 50, in accordance with accepted standards for Good Clinical Practices (GCP) and International Conference on Harmonization (ICH). All subjects provided informed consent prior to study participation.
Subjects were not allowed to apply any other topical products than the ones provided (including skin brighteners, retinoids, alpha/beta/poly-hydroxy acids) nor undergo treatments to the test area. All subjects were randomized either to the NCC-treatment group, which used NCC twice-daily (once in the morning and night, after cleansing) in addition to standard skin care products (SkinMedica® Facial Cleanser and SkinMedica® Essential Defense Mineral Shield SPF 35 Sunscreen), or to the control group, which only used the provided standard skin care products. All subjects were provided with pre-weighed units of assigned products of which they were instructed to apply a thin layer to the neck and décolletage and blend in an upward motion. A total of 75 subjects were enrolled in the study with 46 subjects in NCC-treatment group and 29 subjects in control group.
Clinical Efficacy Assessment
Clinical efficacy was conducted at baseline, weeks 4, 8, and 12 by the investigator who was blinded to the treatment randomization of the subject. Subjects were instructed to cleanse their neck, face, and décolletage and remove all makeup at least 30 minutes prior to each scheduled visit. Clinical assessments and standardized photographs of the neck and décolletage were taken at each visit. Assessments of efficacy parameters were conducted at all study visits using a modified Griffiths’ 10-point grading scale, where 0=none (best possible condition), 1–3=mild, 4–6=moderate, and 7–9=severe (worst condition possible), with half-point scores assigned as necessary to accurately describe the skin condition. Crepiness, laxity/sagging, and overall skin texture were only assessed on the neck and not décolletage. Efficacy parameters are listed below with description of grade 0 and 9 anchors:
Fine lines/wrinkles: 0=none, 9=numerous, long, deep fine lines/wrinkles
Coarse lines/wrinkles: 0=none, 9=numerous, long, deep coarse lines/wrinkles
Crepiness: 0=skin appears smooth with no wrinkles or “crinklinessâ€, 9=skin appears thin and easily crinkled when lightly pinched
Tactile roughness: 0=smooth, even feeling skin texture, 9=rough, uneven feeling skin texture
Laxity/sagging: 0=firm, dense appearance, no sagging, 9=soft, droopy appearance, prominent skin folds
Overall skin texture: 0=smooth skin appearance and touch no roughness, laxity, nor crepey/crinkled appearance, 9=pronounced, extensive visible skin roughness, lines/wrinkles, laxity and crepey/crinkled appearance
mRNA was extracted from the human skin model tissues (Maxwell® RSC simplyRNS Tissue Kit; Promega) followed by cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit; ThermoFisher Scientific) and quantitative real-time PCR (TaqMan Fast Advanced Master Mix, ThermoFisher Scientific). Gene expression analyses were performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) with real-time PCR system QuantStudio7 Flex (ThermoFisher Scientific). The assay mix used for studies were: GAPDH (Cat#: Hs02758991_ g1), COL1A1 (Cat#: Hs001640004_m1); COL3A1 (Cat#: Hs00943809_m1), COL6A1 (Cat#: Hs01095585_m1), COL7A1 (Cat#: Hs00164310_m1), TGFB1 (Cat#: Hs00998133_m1), DCN (Cat#: Hs00754870_s1), ELN (Cat#: Hs00355783_m1), FBN1 (Cat# Hs00171191_m1), FBLN5 (Cat#: Hs00197064_m1 ), MFAP1 (Cat#: Hs00195537_m1), LOXL1 (Cat# Hs00935937_m1), POMP (Cat#: Hs01106088_m1), PSMB5 (Cat#: Hs00605652_m1), PSMB6 (Cat#: Hs00382586_m1), ATG5 (Cat#: Hs00169468_m1), ATG7 (Cat#: Hs00893766_m1), ATG12 (Cat#: Hs04980076_s1), BECN1 (Cat#: Hs01007018_m1), HSPA8(Cat#: Hs03044880_gH).
Elastase Assay
The assay employed was based on methods previously described in literature.8 In brief, human skin model tissue was homogenized and solubilized in 0.1% Triton-X 100, 0.2 M Tris– HCl (pH 8.0) buffer, followed by ultrasonication and subsequent centrifugation at 2000 x g for 10 minutes to obtain supernatants for enzyme assay. To measure elastase activity, 2 μL of substrate (62.5mM, N-succinyl-tri-alanyl-p nitroanilide from Sigma-Aldrich) was incubated with 100 μL lysate solution at 37°C for 2 hours. The amount of released nitroaniline was measured by determining absorbance at 410 nm using a spectrophotometer (Molecular Devices).
Clinical Study Design
Study Design
A randomized, double-blind, regimen-controlled study was conducted to assess the efficacy and tolerability of NCC as a topical treatment for the neck and décolletage. Criteria for study participation included female subjects aged 45–70 years, presenting moderate to severe overall skin texture on the neck and moderate to severe skin tone unevenness on the décolletage.
Institutional Review Board approval (IntegReview IRB, Austin, TX) was obtained prior to conduct of any study procedures.The conduct of the study followed all applicable guidelines for the protection of human subjects for research as per 21 CFR 50, in accordance with accepted standards for Good Clinical Practices (GCP) and International Conference on Harmonization (ICH). All subjects provided informed consent prior to study participation.
Subjects were not allowed to apply any other topical products than the ones provided (including skin brighteners, retinoids, alpha/beta/poly-hydroxy acids) nor undergo treatments to the test area. All subjects were randomized either to the NCC-treatment group, which used NCC twice-daily (once in the morning and night, after cleansing) in addition to standard skin care products (SkinMedica® Facial Cleanser and SkinMedica® Essential Defense Mineral Shield SPF 35 Sunscreen), or to the control group, which only used the provided standard skin care products. All subjects were provided with pre-weighed units of assigned products of which they were instructed to apply a thin layer to the neck and décolletage and blend in an upward motion. A total of 75 subjects were enrolled in the study with 46 subjects in NCC-treatment group and 29 subjects in control group.
Clinical Efficacy Assessment
Clinical efficacy was conducted at baseline, weeks 4, 8, and 12 by the investigator who was blinded to the treatment randomization of the subject. Subjects were instructed to cleanse their neck, face, and décolletage and remove all makeup at least 30 minutes prior to each scheduled visit. Clinical assessments and standardized photographs of the neck and décolletage were taken at each visit. Assessments of efficacy parameters were conducted at all study visits using a modified Griffiths’ 10-point grading scale, where 0=none (best possible condition), 1–3=mild, 4–6=moderate, and 7–9=severe (worst condition possible), with half-point scores assigned as necessary to accurately describe the skin condition. Crepiness, laxity/sagging, and overall skin texture were only assessed on the neck and not décolletage. Efficacy parameters are listed below with description of grade 0 and 9 anchors:
Fine lines/wrinkles: 0=none, 9=numerous, long, deep fine lines/wrinkles
Coarse lines/wrinkles: 0=none, 9=numerous, long, deep coarse lines/wrinkles
Crepiness: 0=skin appears smooth with no wrinkles or “crinklinessâ€, 9=skin appears thin and easily crinkled when lightly pinched
Tactile roughness: 0=smooth, even feeling skin texture, 9=rough, uneven feeling skin texture
Laxity/sagging: 0=firm, dense appearance, no sagging, 9=soft, droopy appearance, prominent skin folds
Overall skin texture: 0=smooth skin appearance and touch no roughness, laxity, nor crepey/crinkled appearance, 9=pronounced, extensive visible skin roughness, lines/wrinkles, laxity and crepey/crinkled appearance
