pharmaceutics Article The Influence of Increasing Concentrations of AMPD on the Efficacy of Its Penetration into a Model Skin Sebum Layer Agnieszka Kostrz˛ebska and Witold Musiał * Department of Physical Chemistry and Biophysics, Faculty of Pharmacy, Wroclaw Medical University, ul. Borowska 211A, 50-556 Wrocław, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-71-784-02-28 Received: 22 September 2020; Accepted: 15 December 2020; Published: 18 December 2020 Abstract: Alcoholamines are widely used as auxiliary substances in various topical preparations. Their impact on the components of skin sebum allows them to be used in preparations that cleanse the skin of sebum in hair follicles. We measured the effects of various concentrations of aqueous solutions of AMPD (2-amino-2-methyl-1,3-propanediol) on model skin sebum. The volume of reacted sebum was calculated using two methods: optical assessment of the interaction of alcoholamines with the components of model skin sebum and determination of the reacted volume of model skin sebum based on the measurements of changes in the pH of the AMPD solutions. Both methods showed that the most favorable AMPD concentration for model sebum penetration was approximately 1–2%. Lower values of alcoholamine caused premature exhaustion from the solution. Excessively high concentrations resulted in the formation of a dense layer of products hindering effective skin cleansing. Keywords: skin sebum; alcoholamines; 2-amino-2-methyl-1,3-propanediol; AMPD; acne 1. Introduction Acne vulgaris is one of the most common skin diseases of adolescents and young adults. It is characterized as a sebum-producing sebaceous gland disorder. The main pathophysiology of acne involves abnormal exfoliation of epidermal cells, excessive sebum secretion, and the action of bacteria that colonize hair follicles [1]. As a result of the activity of bacteria of the human microbiome, such as Propionibacterium acnes, P. granulosum, Staphylococcus epidermidis, and S. aureus, there is the formation of sebaceous metabolic products including free fatty acids and other pro-inflammatory substances that cause hair follicle clogging [2–5]. For local acne treatment, antibacterial, hygiene, and care products are used most often. They work by removing the surface layer of lipids from the skin. Usually, however, they do not cleanse clogged sebaceous glands [6–8]. The proposed mechanism for cleansing sebaceous glands more thoroughly may be the binding of free fatty acids from sebum with alcoholamines to form amine soaps and easier removal of sebum from glands. [9–11]. Alcoholamines are characterized by emulsifying, thickening, foaming, and preserving properties. Their alkaline nature is used to regulate the pH of cosmetics. Many of them, including AMPD (2-amino-2-methyl-1,3-propanediol), MEA (monoethanolamine), DEA (diethanolamine), TEA (triethanoloamine), TRIS (tromethamine), MIPA (monoisopropanolamine), and TIPA (triisopropanolamine), are used in the cosmetics and pharmaceutical industries as excipients. They are used in the production of creams, ointments, shampoos, emulsions, lotions, and tonics. They are also widely used in pharmaceutical technology as auxiliary substances and derivatives for the production of medicines [12–14]. Pharmaceutics 2020, 12, 1228; doi:10.3390/pharmaceutics12121228 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 1228 2 of 14 Previous studies have demonstrated the reactions between various alcoholamines and the components of the model sebum, leading to the formation of amine soap. The resulting soap binds water due to the presence of hydroxyl groups in the alcoholamine molecule. It stabilizes the O/W emulsion, increases the volume of sebum in the gland, and loosens it. This action facilitates the elimination of sebum from the gland. Together with sebum, P. acnes, which is responsible for acne inflammation, can be removed [9–11]. It is also possible to create alcoholamine complexes with various additives such as anionic polymers. These affect the prolongation of the action of alcoholamines and reduce the alkalinity of the system without affecting the reaction of alcoholamines with model sebum [10,11]. Based on previous studies and our observations of selected alcoholamines, we chose AMPD for further research. Its molecular weight is 105.14, pKa = 8.76 at 25 ◦C, it is soluble in water and alcohol [13]. We prepared aqueous solutions of AMPD with concentrations of 0.5% to 2.5%. The pH of these solutions did not exceed 11, which places AMPD in the middle of the alcoholamines selected for this study. Then we placed them on the model sebum layer, observing the changes for 4 h. We also carried out a 4-h measurement of changes in the pH value over the sebum in the AMPD solution. AMPD activity with respect to the components of model sebum was superior to that of other alcoholamines. AMPD penetrated well into the model sebum. The swelling of the ingredients over the sebum layer was also effective. 2. Materials and Methods 2.1. Reagents The following reagents were used as model skin sebum components: triglycerides of animal origin (Cefarm, Warszawa, Poland), stearic acid (Sigma-Aldrich, Poznan, Poland), squalen (Sigma–Aldrich), cholesterol (Sigma–Aldrich), and lanolin (Cefarm). The following alcoholamines were selected as substances penetrating model skin sebum: AMPD (Sigma-Aldrich), TRIS (Sigma-Aldrich), DIPA (Sigma-Aldrich), and TIPA (Sigma-Aldrich). Demineralized and bi-distilled water were used to form aqueous solutions of alcoholamines. 2.2. Analytical Methods 2.2.1. Composition and Preparation of Model Skin Sebum The composition of a model skin sebum was based on the available literature. The skin sebum is a mixture of lipids that differ in chemical properties from biological membranes. Its composition includes triglycerides, squalene, cholesterol, and waxes [15–17]. Based on previous studies, model sebum was prepared to contain pork lard for triglycerides (34%), stearic acid as a free fatty acid (24%), lanolin as wax (26%), squalene (12%), and cholesterol (4%) [10,11]. This sebum formulation, developed by Kubis and Musiał, was recognized by Stefaniak and Harvey as containing important lipid classes with constituents at concentrations corresponding with human sebum including squalene, wax esters, triglycerides, free fatty acids, and free cholesterol [18]. The ingredients were melted and mixed in a water bath and left to solidify. 2.2.2. Determination of Model Skin Sebum Density The density of model skin sebum was determined using a pharmacopoeial method using a pycnometer [19]; 25% of the pycnometer volume was filled with molten model skin sebum and was then weighed after cooling to 20 ◦C. The volume was topped up with 20 ◦C water, and the sample was weighed again. Calculations were made based on Equation (1) [19]: m d = 1 0.997 + 0.0012 (1) w + m m × 1 − 2 Pharmaceutics 2020, 12, 1228 3 of 14 where d is the density of model skin sebum, m1 is the mass of skin sebum, w is the mass of pycnometer with water and m2 is the mass of pycnometer with model skin sebum and water weighed at 20 ◦C. The value 0.997, given in the equation, is the density of water at 20 ◦C, and 0.0012 is a correction for air weighing. The density of model skin sebum used in the study was 0.842 mg/mm3. This value is similar to the sebum density of healthy people and people with acne (0.9 mg/mm3 0.01) [20]. ± 2.2.3. Optical Assessment of the Interaction of Alcoholamines with the Components of Model Skin Sebum To determine the volume of reacted sebum, the height of the reacted sebum was measured in polystyrene tubes of 16 mm in diameter. A 2 cm3 piece of sebum was placed in the tube, on which a 3 cm3 aqueous AMPD solution was applied. The course of the reaction was observed for 4 h at strictly defined intervals. The exact height of the reacted sebum layer was determined using digital macro photography, following the rule applied in the research of Kubis et al. [10,11]. We used a Fuji FinePix S6500FD digital camera (Fuji Photo Film Co., Ltd., Tokyo, Japan), 6.3 megapixels, with a 6.2–66.7 mm variable focal length lens, as well as an original program for digital image processing. 2.2.4. Measurement of the Degree of Turbidity of the Evaluated Solution above the Sebum Layer The turbidity of the evaluated solution over model sebum was measured using a single-beam spectrophotometer (Spekol 11, Carl Zeiss, Jena, Germany) using a TK1 attachment. Small particles can scatter short wavelengths of light more efficiently comparing to long wavelengths, which may result in disproportionately high measurement. To compensate for this effect during the measurements, we decided to use a light length close to the IR range (780–900 nm) [21]. The maximum wavelength, at which the measurements were carried out, was determined experimentally based on a series of diluted suspensions resulting from the highest concentration of AMPD with sebum components. This value was 850 nm. After four hours of observation, the solution with 0.5% AMPD showed the highest degree of turbidity in the solution. The turbidity of the evaluated solutions above the layer of model skin sebum decreased as the concentration of the AMPD increased. 2.2.5. Measurement of Changes in the pH Value of AMPD Solutions over a Layer of Model Skin Sebum Changes in the pH of the alcoholamine solution above the model skin sebum, associated with the reaction of amine soap formation, were measured using a Hanna Instruments pH302 (Hanna Instruments, Woonsocket, RI, USA) with a suitable computer program. pH measurements were taken at one-minute intervals for four hours. We applied 3 g of AMPD solutions to a 2 cm thick model skin sebum layer placed in a tube with a diameter of 16 mm. The tubes were protected against air by paraffin film.