Patient recruitment and trial design
This prospective, randomized, placebo-controlled, exploratory study was conducted to test the hypothesis that a topical application of rapamycin (10 μM) would decrease the number of senescent cells in the skin and decrease markers of aging. Subjects were recruited at the Drexel University College of Medicine Department of Dermatology in Philadelphia, PA. The study protocol was approved by the Drexel University Institutional Review Board and was conducted in accordance with ethical principles outlined in the Declaration of Helsinki. Participants were greater than 40 years of age and had no history of diabetes or hypercholesterolemia. Full inclusion and exclusion criteria are included in the study protocol provided in Supplemental Materials. Participants enrolled in the study were provided with a container of rapamycin cream and a container of placebo (DMSO) in identical dispensers with labels for right or left hand and were instructed to apply the creams 0.5 cc (1 pump from the dispenser) to the dorsal side of each hand every 24 to 48 h in the evening before bed. Participants were evaluated at follow-up visits 2, 4, 6, and 8 months after initiation of the topical application. Wrinkles and clinical appearance of the skin were documented by photography at each clinic visit. Blood was drawn from consenting individuals (n = 13) at the 6-month visit to assess systemic delivery of rapamycin, and tissue from the dorsal surface of both hands was obtained by biopsy at final visit, using a 3-mm punch probe. Biopsies were divided into 2 sections for further analyses (histological evaluation, immunohistochemistry, isolation of total RNA). Rapamycin was measured in whole blood by an independent CLIA-certified laboratory (NMS Laboratories, Horsham, PA).
Clinical assessment of dermal tissue
Clinical signs of aging were evaluated at each study visit. The appearance of the dorsal hands was evaluated using the Merz Hand Grading Scale, a validated measure where clinical hallmarks of aging such as prominent veins and tendons are associated with a higher score (Cohen et al. 2015). Fine wrinkles were assessed using an internal grading scale consisting of the following metrics: 0—absent, 1—slight, 2—evident, 3—marked, 4—very marked. Dyspigmentation and skin tone were evaluated using the Glogau Classification of Photoaging, modified to exclude assessment of facial wrinkling (Durai et al. 2012).
Biopsies were fixed in 3% formalin for > 24 h, washed with PBS, and processed by the Pathology Diagnostics Laboratory in the Department of Pathology and Laboratory Medicine at Drexel University College of Medicine. Samples were processed and embedded in paraffin for sectioning. Immunohistochemistry was performed using the Ventana XT Ultra system (Roche Diagnostics) using the ultraView DAB detection kit. Antibodies and staining protocols were as follows: p16INK4A (Enzo Life Sciences, ABS377-1000), p21Cip1/Waf1 (Cell Marque, DCS-60.2), tp53 (Ventana, D0-7), and cytokeratin 5/6 (Ventana, DS/16B4), with the exception of collagen VII which was a rabbit polyclonal antibody obtained from Abcam (ab93350). Antigen retrieval was performed using citrate buffer (36–64 min) except for collagen VII, which utilized enzymatic digestion (protease for 64 min). Blocking was performed for 24–32 min, and antibody incubation times ranged from 24 to 60 min. Antibodies and staining conditions are reported in Supplemental Table 1.
RNA isolation and analysis
Total RNA was isolated from tissue biopsies using Qiagen RNA easy protocols. Quantity and integrity of RNA were initially evaluated using a NanoDrop 2000/2000C spectrophotomer (Thermo Fisher) and confirmed using fluometric analysis on a Qubit fluorimeter (Thermo Fisher). Finally, integrity was evaluated using a Bioanalyzer RNA 6000 Nano assay (Agilent). Expression analysis was performed using the nanoString nCounter expression system housed in Drexel University College of Medicine Department of Microbiology and Immunology. Gene expression analysis was performed using a custom gene expression panel that included collagen, keratin isoforms, inflammatory cytokines, and senescence-associated genes. Levels for two primary senescence-associated gene products, p16INK4A and p21Cip1/Waf1, were below the level of detection in the tissue extract likely due to a low level of senescent cells relative to total tissue. Normalization was performed according to default settings in the nSolver software, and differential expression created using ratio versus rapamycin-treated is presented in Supplemental Table 2.
Staining was evaluated as percent positive nuclei (p16INK4A, p21Cip1/Waf1, tp53) or staining intensity (collagen VII). Positive nuclei were scored using an automated cell scoring pipeline developed on the Aperio Systems. Collagen VII staining was scored based on independent visual assessment of immunohistochemical staining on a 1- to 4-point Likert scale. The data appear to follow a normal distribution; however, because of the small sample size, data were evaluated using both parametric and non-parametric testing.