Percutaneous Absorption Study of Progesterone in Freedom HRT Supreme Cream and Freedom HRT Natural Cream Summary The objective of this study was to determine the unidirectional permeability or percutaneous absorption of progesterone in Freedom HRT Supreme Cream and Freedom HRT Natural Cream, in vitro, across frozen split thickness human skin. The permeability assay was determined utilizing Franz diffusion cells. Materials and Methods The two formulations studied were 5% Progesterone in Freedom HRT Natural Cream and 5% Progesterone in Freedom HRT Supreme Cream and were provided by Freedom Pharmaceuticals. Atenolol, testosterone, sodium azide, and Krebs-Ringer bicarbonate (KRB) buffer mix were obtained from Sigma-Aldrich. KRB buffer was prepared according to the manufacturer’s instructions, supplemented with 1.2 mM CaCl2, 15 mM NaHCO3, and 10 mM HEPES, and was adjusted to a final pH of 7.4. Static, vertical glass Franz diffusion cells were used for the permeability assay. Donor Demographic Dermatomed skin from a post-mortem human donor was obtained from National Disease Research Institute. Gender: Male / Race: Caucasian / Age: 56 / Cause of Death: Cardiac Arrest Formulations Progesterone 5% in HRT Natural Cream Progesterone USP Ultra Micronized . . . . . . . . . . . . . . . . . 5% Ethoxy Diglycol Reagent . . . . . . . . . . . . . . . . . . . . . . . . . 5% Freedom HRT Natural Cream . . . . . . . . . . . . . . . . . . . . . 90% Progesterone 5% in HRT Supreme Cream Progesterone USP Ultra Micronized . . . . . . . . . . . . . . . . . 5% Ethoxy Diglycol Reagent . . . . . . . . . . . . . . . . . . . . . . . . . 5% Freedom HRT Supreme Cream . . . . . . . . . . . . . . . . . . . . 90% Permeability Experiments The dosing solution with control compounds (atenolol and testosterone, 100 μM each) was prepared in KRB buffer at pH 7.4 on the same day, immediately prior to the start of the permeability experiments. The skin was kept frozen at -80°C until the time of the study. The skin was soaked in saline for 30 minutes at room temperature (RT) to thaw, cut to appropriate size (~2×2 cm). The thickness of the tissue was measured for each piece and sorted. They were grouped randomly to ensure the average thickness is approximately similar. Tissue was mounted into the Franz-cell diffusion chamber and clamped between the donor and receiver chambers. The exposed surface area of the Franz-cell diffusion chamber was 1.77 cm2. The receiver compartment was filled with 8 mL KRB buffer containing 1% BSA and 0.1% sodium azide. The reservoir also contained a stirring bar stirring at 400 RPM to ensure the homogeneity of the reservoir contents. Each Franz-cell diffusion chamber was placed in a dry block heating/stirring module with temperature set to maintain the tissue surface at approximately 32°C. freedomrxinc.com • 877.839.8547 Percutaneous Absorption Study of Progesterone in Freedom HRT Supreme Cream and Freedom HRT Natural Cream Permeability Experiments - cont. Progesterone Flux (µg/cm2/hr) at Time Approximately 50 mg of each formulation, which equates to 61.5 μl of 5% Progesterone in Freedom HRT Natural Cream and 55 μl of 5% Progesterone in Freedom HRT Supreme Cream, was transferred into donor chambers using a positive displacement pipette. 2 mL of control solution was added into the donor chamber of the Control group. Samples (1 mL) were taken from the receiver compartment at 2, 4, 8, 24, 30 and 48 hours. After each sampling, fresh buffer of an equal volume was added to replace the buffer removed to maintain sink condition. The donor compartment of the Control group was sampled at the onset and end of the assay. At the end of the permeability assay, skin tissue treated with the above named formulations was removed from the chambers, wiped off excess test materials, rinsed in saline, weighed and stored at -80°C until analysis. 0.25 Flux (µg/cm2/hr) 0.2 0.15 0.1 0.05 0 Unidirectional Permeability across Human Skin The results indicated the progesterone flux was comparable in formulations made with both Freedom HRT Natural and Freedom HRT Supreme Cream. 0h 2h 4h 8h HRT Natural 24h 30h 48h HRT Supreme Overall Progesterone Penetrated Across Stratum Corneum % Applied Progesterone that Penetrated Stratum Corneum The overall percentage of progesterone penetrated across stratum corneum was statistically comparable in formulations made with both Freedom HRT Natural and HRT Freedom Supreme Cream. % Applied that Penetrated S.C. 2.0 Conclusion Progesterone from both formulations tested, permeated across human skin samples at a similar and consistent rate. There was no statistically significant difference between formulations in terms of flux values, or percentage of applied progesterone that penetrated stratum corneum. This conclusion suggests that formulations in Freedom HRT Natural Cream and Freedom HRT Supreme Cream demonstrate favorable percutaneous absorption of Progesterone. 1.5 1.0 1.323 0.5 0.977 0.0 HRT Natural HRT Supreme freedomrxinc.com • 877.839.8547
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