2.1. Ointment pH Measurements by the Potentiometric Method
Table 1 shows the results of the pH measurements. Measurements were taken for Lekobaza
® (F-1) itself, for Lekobaza
® with the addition of 1 mol/L aqueous acetic acid solution (F-2), and for ointments with corticotropin with the following concentrations: 5 mg ACTH/g ointment (F-3), 10 mg/g (F-4), 15 mg/g (F-5), 20 mg/g (F-6), and 25 mg/g (F-7).
The highest value of pH (6.30) was noted for Lekobaza
®. The addition of 1 mol/L acetic acid solution significantly reduced the pH to 3.43. Corticotropin added to the ointment, depending on its concentration, caused in turn a slight increase in the pH value in comparison with the ointment with the 1 mol/L acetic acid solution only. The more corticotropin in the ointment, the higher the pH value. The lowest pH value was the ointment with ACTH 5 mg/g (pH = 3.61), and the highest pH value was the ointment with ACTH 25 mg/g (pH = 4.00). Some excipients may increase the pH of the skin, causing skin barrier damage [
25], while other ingredients may have a beneficial effect by lowering the pH of the skin [
26] and protecting the acidic skin coat. For example, the inborn antimicrobial properties of the skin are optimal for acidic pH, because the
Staphylococcus and other pathogenic bacteria promote a neutral pH and are inhibited in an acidic environment [
27]. Moreover, in an acidic environment, the correct exfoliation of the stratum corneum is a process regulated by the kallikrein enzymes “KLK5” and “KLK7” [
28]. However, at higher pH, skin cell flaking may get out of control, damaging the stratum corneum barrier [
25,
26,
27,
28,
29].
Corticotropin is an alkaline substance. In an acidic environment, such as a 1 mol/L acetic acid solution, it is possible to increase the stability of this peptide, which remains in the salt solution, acetate. ACTH is dissolved in 0.1 mol/L acetic acid, but acetic acid of 1 mol/L was used to enhance this effect. During corticotropin extraction from the pituitary glands, the extraction takes place in an acidic environment, in order to make the penetration of this hormone into the solvent more effective. The pH value was determined in the tested ointments, and no pH modification was introduced here yet, as these were preliminary studies. However, this modification requires testing of the stability of ACTH by increasing the pH.
2.2. Spreadability of the Ointments
In order to evaluate the effect of the addition of an aqueous solution, two formulations were compared. One formulation was a control and it was Lekobaza
® only, and the second formulation was Lekobaza
® with the addition of a 10% aqueous acetic acid solution of 1 mol/L. In order to present the effect of the ACTH content on the ointment spreadability, the course of spreadability curves were compared with this parameter for the control ointment with the acetic acid solution (F-2). The course of the spreadability curves is shown in
Figure 1.
Lekobaza® is a base with good spreadability in the assessment of the manufacturer (Fagron, Kraków, Poland). The assessment of the spreadability of a topical or mucosal semi-solid preparation is influenced by the hardness or hardness of the composition, shear rate and time produced after smearing, and temperature at the target site. The spreading efficiency also depends on the formulation viscosity, solvent evaporation rate, and evaporation rate and intensity, with an increase in viscosity along with evaporation.
The addition of 10% aqueous solution to Lekobaza® (F-1) makes the spreadability of Lekobaza® with acetic acid solution (F-2) even higher (p < 0.05). However, with an increase in the concentration of ACTH introduced into Lekobaza® as a solution in 1 mol/L acetic acid, the spreadability (p < 0.05) decreased in comparison with the F-2 preparation (Lekobaza® with the addition of 10% aqueous solution). It proves that with an increasing concentration of ACTH, the spreadability will deteriorate.
It was observed that the ointment with the highest ACTH content (25 mg/g) showed the lowest spreadability and the ointment with the lowest ACTH concentration (5 mg/g) showed the best spreadability. Ointments with concentrations of 10 and 15 mg/g, F-4 and F-5, showed worse spreadability than F-3 (5 mg/g) but better than F-7 (25 mg/g). The exception is a formulation with a concentration of 20 mg/g (F-6).
The highest concentration of ACTH, 25 mg/g, reduces the spreadability to the greatest extent, followed by 10, 15, 20, and 5 mg/g (
p < 0.05). The spreadability of the 10 mg/g ACTH ointment did not differ significantly from this parameter for the control ointment (F-1).
Table 2 shows the parameters describing the ointment spreadability, including equations describing the course of the spreadability curves, correlation coefficients (R
2), area under the spreadability curves, and the spreadability index (i(S)) in relation to the control ointment F-1. It was found that when i(S) is greater than 1.0, the tested ointment has better spreadability, and when it is less than 1.0, the ointment has worse spreadability in relation to the F-1 formulation.
Therefore, only ointments F-7 and F-4 have weaker spreadability in relation to F-1. If one assumes that Lekobaza
® has good spreadability, ointments with ACTH also show proper spreadability: F-3, F-5, and F-6. This is compensated by the addition of a water solution. For the tested ointments, the area under the spread curve (AUC) was calculated, and a weak negative correlation was found for the area under the curve (AUC [cm
2]), depending on the ACTH concentration in the ointment (R
2 = −0.428). Similar results were obtained by Szulc-Musioł et al., who found an inverse correlation between the spreading rate and quercetin content (R
2 = −0.283,
p ≤ 0.05) [
30].
The rheological features of the ointment—plasticity, flow properties, thixotropy—enable the preparation, spreading, and packing of the ointment. Change of viscosity by shear stress allows spreading of the ointment on the skin, and prevents the ointment from flowing out of the skin. Pressure on the ointment tube creates a certain shear stress, which causes the system to flow. It allows the ointment to be squeezed out of the tube. The appropriate flow limit prevents the ointment from accidentally flowing out. When the ointment is mixed (as well as during heating), its organized internal structure is disturbed. The structure can be restored to its original state by rebuilding it in order to maintain the physical stability of the system.
In this work, it was expected that the addition of the drug would not deteriorate the spreadability parameter in relation to the base itself, which is characterized by good spreadability and an appropriate consistency. After application of the preparation on the skin, the drug will last for a certain period of time and will ensure the longest possible contact with the drugs without the risk of the preparation flowing out.
2.3. Rheological Properties
Viscosity is not a constant value and depends on factors, such as the temperature and shear rate. The storage and application temperature of the preparation can be used for measurements. The range of shear rate can also be selected based on the storage of the ointment or the technique of spreading it on the skin. The rheological tests in references were carried out at 20–37 °C. In our study, the viscosity, flow step test, and flow test were tested at a controlled shear rate at two temperatures, 25 and 32 °C. The resting body temperature is approximately 37 °C and the weighted average skin surface temperature is between 32 and 34 °C. This temperature corresponds to the application of ointment on the skin surface. The temperature of 25 °C corresponds to the room temperature at which the ointments are normally stored, just before its application to the skin. Three shearing rates: 300, 700 and 1100 s
−1 were used in the tests. The determined values of viscosity and shear stress are presented in
Table 3.
The temperature and shear rate significantly affect the viscosity of the formulation. At shear rates of 300 and 700 s−1 at 32 °C for formulations without active substance and containing low concentrations of ACTH (5 and 10 mg/g), the viscosity is reduced 2 to 2.5 times. For higher concentrations of ACTH, on the other hand, viscosity increases slightly, about 1.1 times under the same conditions. At the wall speed of 1100 s−1 with a temperature increase, viscosity decreases about 1.5 times for F-1–F-5 formulations, while for F-6 and F-7, it practically does not change, and is not statistically significant. Decreasing the viscosity of the formulation during spreading of the preparation on the skin may contribute to the increase of the active substance’s release from the ointment.
The addition of 10% aqueous solution to Lekobaza® in a weight ratio significantly decreased the viscosity, 1.2 times for almost all preparations to 2 times for F-1 (control ointment). This may result from thinning of the base as an emulsion system is formed.
The active substance added to the ointment increased the viscosity depending on the hormone concentration in the ointment. This relation is best visible in comparison with hydrated Lekobaza® F-2. The smallest increase in viscosity was observed at a concentration of 5 mg/g ACTH at 25 °C and it increases as follows: F-2 < F-3 < F-4 < F-5 < F-7 < F-6. The smallest increase in viscosity was observed at a concentration of 10 mg/g ACTH at 32 °C and it increases as follows: F-4 < F-5 < F-7 < F-6. The preparation at a concentration of 20 mg/g ACTH caused the highest increase in viscosity.
To prevent excessive viscosity increases at higher corticotropin concentrations, ACTH is included as an aqueous solution to create an emulsion instead of a suspension ointment. It is possible to improve the physicochemical properties of the formulations, including their spreadability, pharmaceutical availability, and bioavailability, and thus improve the effectiveness of the semi-solid drug forms. In the case of the cream base Lekobaza
®, it is possible due to its ability to absorb a significant amount of water (water number > 300). Kolpakova et al. (2019) tested ointments, such as emulsions, with a water-soluble protein-polysaccharide complex. It was found that all the tested ointments were visco-structural systems, and the ointment composition, complex concentration, and character of the emulsifier appropriately selected by the formulator provided the necessary thixotropic properties [
31].
The rheological parameters of the ointment with different concentrations of ACTH were examined in the study. The hydrophilic medium of a cream character, Lekobaza®, was used as an ointment base. ACTH was introduced into the ointment as an aqueous solution in acetic acid. It could be expected that the aqueous solution would decrease in the ointment viscosity and thus increase its spreadability. However, with the increase in the ACTH concentration in the ointment, an increase in viscosity was observed respectively, with the exception of the 20mg/g concentration. An increase in the ACTH concentration from 0.5% to 2.5% for the two shear rates is accompanied by an increase in viscosity. An exception is the ointment with a concentration of 2%, and the causes of this phenomenon may be different. Corticotropin is alkaline in nature and may react with acidic components of the base; perhaps at a concentration of 2%, the effects are more intense. It can also be affected by a charge coming from one of the base components.
Lekobaza
® contains cetyl alcohol, which also increases the melting point of some ointment bases and suppository media, so it may increase the viscosity of the preparations. Cetyl alcohol also acts as a non-ionic water/oil emulsifier in emulsions. The tested ointments showed a weak negative correlation between the spreadability and viscosity (R
2 = −0.293 at 25 °C and R
2 = −0.281 at 32 °C). In turn, Vennat, Gross, and Pourrat, showed the existence of a strong negative correlation between the spreadability and viscosity of elastomeric materials [
32,
33]. The tested ointments are diluted shear systems. The formulations are shear thinning fluids, and their properties are confirmed by the shape of the viscosity curves in
Figure 2A,B. Such properties of Lekobaza
® at 25 °C and Lekobaza
® with a 20% water content at 32 °C were also demonstrated by Szulc-Musioł et al. and Tal-Figiel et al. [
30,
34].
The relationship between the shear stress and shear rate is presented in
Figure 3A,B. The course of the flow curves of all prepared ointments indicated a non-Newtonian character. The relationship between shear stress and shear velocity deviated from a straight line, and there was also an offset along the ordinate in the shear rate range from 100 to 1100 s
−1. Therefore, the equation for non-linear viscoelastic bodies, Casson’s Equation (1), was used to approximate the values of the shear stress and shear rate:
where
τ = shear stress,
τy = constant interpreted as yield strength,
η = constant, and
γ = strain rate (shear rate). This method allows the determination of the flow limit, and its existence is indicated by the shape of the flow curve [
35].
The viscosity of a thixotropic system is dependent on both the shear rate and time. During the process, the structure of the viscoelastic body can be destroyed, and thus its viscosity can be reduced, and its structure can be restored, and its viscosity can increase. Both of these phenomena are significant both from the point of view of the technological process and the application of the finished product to the place of action, the skin. During preparation and mixing, the ointment must lower its viscosity whereas a heightened viscosity is more appropriate for storage or application. High viscosity prevents sedimentation of undissolved particles in the base and movement of the ointment on the skin surface. This would make it difficult for the preparation to act in a specific place (depth of penetration or transport through skin differs due to the thickness of various regions of the skin).
The most suitable method of thixotropy measurement is to describe the material response in shear stress caused by the given deformation or a shear rate [
35]. The shear rate increased with time until the maximum shear value was reached. This process is then reversed by decreasing the shear rate, leading to the up/down curves. The area enclosed by the up/down curve is called the hysteresis loop. In these studies, all formulations show thixotropic properties. This is evidenced by the shape of the loop, as the ascending curve is above the descending curve [
35].
Hysteresis loops of F-2–F-7 at a temperature of 25 °C are shown in
Figure 4A-1–4A-7 and the hysteresis loops of F-2–F-7 at a temperature of 32 °C are shown in
Figure 4B-1–4B-7. Formulation F-1 is shown as the control ointment. At 32 °C, the hysteresis loops are smaller than at 25 °C, and lower values of their surface were observed.
Formulations of incompatible compositions tend to produce a structure that is destroyed at high shear rates and which undergoes reforming during ageing at elevated temperatures or excessive pH [
35]. It has been reported that the viscosity of hydrogels deteriorates after the gelling agent is roasted and the microcrystalline cellulose (MCC) concentration increases [
36]. Moreover, the character of the gelling agent also influences the viscosity parameter: Too high a degree of swelling decreases the degree of swelling and viscosity of the gel formed [
37]. In our study, we observed that the addition of an aqueous solution changed the structure of the ointment, similar to the addition of the drug. Perhaps the opposite effect would be due to the addition of more water to the emulsion instead of 10% (e.g., 20%). The smallest changes were caused by 5 and 10 mg/g ACTH.
Based on the hysteresis loops, it can be concluded that, in the case of ointments with a concentration of 5 mg/g (F-3) and Lekobaza® with acetic acid (F-2), the loops overlapped and the ability to recover after deformation was similar for these formulations. The ointment with the highest ACTH content, 25 mg/g, should seem to have the largest surface area, although from the viscosity measurement at three shear rates, it turned out that the ointment with the concentration of 20 mg/g has the highest viscosity, so it can be expected that the formulation at the concentration of 20 mg/g ACTH showed the lowest rheological stability. Based on the significant difference between the initial and final viscosity values during the flow test, which do not overlap at all, the preparation at a concentration of 20 mg/g ACTH can be expected to show the lowest rheological stability. It can be concluded that with the increase of the ACTH content in the ointment, its structure requires more time to return to the original structure: The ascending curve becomes more and more distant from the descending curve. Therefore, the course of viscosity in the range from 100 to 1100 s-1 reflects thixotropy better than single selected shear rates values. The flow test allows the rheological stability of the formulation to be evaluated.
In comparison, the slope of the non-linear parts of the curves of all preparations at 32 °C is slightly milder than at 25 °C, and the ascending and descending curves are closer together at 32 °C. As the concentration of ACTH increases, the distance between the ascending and descending curves increases. Rheograms received during tests at two temperatures, 25 and 32 °C, show slight changes of the shape of the overall rheogram as the temperature increases. At 32 °C, a decrease in the distance between the ascending and descending curves was observed for each formulation, which is the result of lower shear stress due to the decrease in viscosity at this temperature. Regarding the point of view of the topical application of the preparation, it is a beneficial phenomenon for spreading the preparation on the skin because the temperature of 32 °C corresponds to the temperature on the skin surface.
2.4. Texture Analysis
The “TPA Cycle” (texture profile analysis) allows for the performance of a standardized TPA cycle that includes two compression/stretching cycles with a pause between them. These two cycles allow the determination of the hardness, adhesion, elasticity, and cohesion based on the A2/A1 ratio of the two compression phases. Material hardness is defined as the maximum force recorded during the first phase of compression. The value of F
min measured in the first phase of stretching is defined as the adhesion force. Adhesion is the work during the first phase. Adhesion is the work needed to overcome the forces of attraction between the ointment surface and the probe surface (work necessary for detachment of the probe from the sample) [
38]. The D2/D1 ratio between two compression phase distances is flexible. The values of the marked parameters are presented in
Table 4. Four formulations were selected for the study: Lekobaza
® (F-1) as a reference, Lekobaza
® with an emulsified acetic acid solution (F-2), and two ointment formulations containing the lowest concentration of ACTH (5 mg/g (F-3)) and one of the higher ones of ACTH (20 mg/g (F-6)), to present the effect of the active substance in extreme quantities.
Figure 5 shows an exemplary texture analysis curve of formulation F-3 (5 mg ACTH/g ointment). From the resultant force–time plots, the following mechanical parameters were derived:
Hardness (the force required to attain a given deformation) was found from maximum force Fmax1.
Cohesiveness (the ratio of the area under the force–time curve produced on the second compression cycle to that on the first compression cycle, where successive compressions are separated by a defined recovery period).
Adhesiveness (the work required to overcome the attractive forces between the surface of the sample and the surface of the probe).
Elasticity: The D2/D1 ratio between two compression phase distances.
Adhesion force: In tension, the force is constantly measured and gives a higher negative value F
min and a curve below ‘0′, which characterizes the adhesion of the sample [
39].
As shown in
Table 4, emulsifying 10% aqueous solution into the medium reduces the hardness by 50%, while the addition of ACTH increases the hardness of the ointment. For example, with ACTH 20 mg/g (F-6), the hardness has doubled in comparison with Lekobaza
® (F-1) (
p < 0.05). The aqueous phase added to the ointment base decreased the adhesion (A) of the ointment by about 1.7 times, but ACTH increases this parameter depending on the amount added, from 1.2 times (5mg/g (F-3)) to more than 5 times (20 mg/g (F-6)) (
p < 0.05). The adhesion strength (AF) increased with increasing adhesion. As a result, semi-solid formulations can adhere to the application surface for a sufficiently long time, which may affect the drug’s residence time at the application site [
40].
When developing preparations for skin and mucous membrane applications, the cohesiveness of the ointment components should be considered [
41]. The addition of both water and corticotropin to the tested formulations resulted in decreased cohesiveness. The higher the hormone concentration, the higher the decrease in cohesion (over 3 times (F-6/F-1)) (
p < 0.05). Similar behaviors were observed in the case of elasticity changes, as water and corticotropin reduced this parameter, with a greater decrease in ACTH 20 mg/g (
p < 0.05). The more additional ingredients in the ointment, the more it can worsen the texture properties.
Additionally, it is clearly visible that the composition used in the formulation influenced the strength of the designed ointment formulation (
Figure 6). The tensile strength and Young’s modulus (modulus of elasticity, [Pa]) were calculated using the Equations (2) and (3):
where
F [N] represents the force applied to the ointment,
A [m
2] displays the calculated cross-sectional area of the ointment cylinder, Δ
l [m] represents the length deformation, and
l [m] represents the original sample length. Young’s modulus is a measure of the “stiffness” (mechanical response) of a material, the material’s ability to rebuild its original shape after deformation [
42].
With an increasing concentration of ACTH in the ointment, its strength parameters increase. Tensile strength increases, but also the hardness of the ointment increases. With the increase of the textural parameters, the time of release of the active substance may be prolonged. It may indicate the possibility of preparing preparations with a prolonged release process. The addition of an aqueous solution, 1 mol/L acetic acid solution, reduced the tensile strength of the ointment base (F-1) about 2 times. A small addition of ACTH (5 mg/g ointment—F-3) slightly increased the tensile strength of the ointment (F-3), whereas a 4 times higher concentration of ACTH (20 mg/g) caused a twofold increase in the tensile strength of the ointment preparations (F-6) in comparison with F-1 and 4 times in comparison with F-2.
The spreadability of the ointment is inversely proportional to its cohesion, because its internal structure is strong and durable. Cohesive forces reduce its fluidity, and thus its ability to spread on the ground. Therefore, each formulation must be specially designed according to the desired purpose and place of application. The spreadability of the ointment can be increased or decreased, as per the assumption that it meets certain requirements [
32,
33,
43].
2.5. In Vitro Drug Release Profiles of the Ointments
A USP apparatus 2 with enhancer cells was utilized to determine the in vitro drug release profiles of the ointment formulations. The results showed that ointments containing different concentrations of ACTH had different rates of release from 90 to 150 min.
Figure 7 shows the cumulative amount of released ACTH. ACTH released quicker from the ointments at lower ACTH concentrations (15 and 20 mg/g) and it had a higher drug release rate (90 and 120 min) compared to ointments with a higher ACTH concentration (25 mg/g). In addition, the Higuchi model was employed to calculate the drug release rate of the ACTH ointment formulations (
Table 5). In the present study, the time range used for the Higuchi modeling of release rate was 15 to 150 min. The drug release rate and goodness of fit using the Higuchi model for all the ACTH ointment formulations are listed in
Table 6. All the formulations showed an adequate fit to the Higuchi model since the R
2 values are greater than 0.9.
The Weibull model is more useful for comparing the release profiles of matrix-type drug delivery [
44]. The Weibull method (
p < 0.05) showed significant statistical differences in the release profiles of ACTH from prepared ointments of 20 mg/g—F-6 (R
2 = 0.98) and 25 mg/g—F-7 (R
2 = 0.81) in relation to the F-5 formulation, from which ACTH was released in the shortest 90 min (R
2 = 0.97). Based on the t-student analysis, formulations at a concentration of 25 mg/g of ACTH showed significantly lower (
p < 0.05) drug release rates compared to ointments at concentrations of 20 and 15 mg/g. Based on the Higuchi model, the shortest release time was characteristic for the preparation with the lowest ACTH concentration (15 mg/g-0.41 mg/cm
2/t
1/2), ACTH was released from ointments at concentrations of 20 mg/g at a rate of 0.48 mg/cm
2/t
1/2, and ACTH was released from ointments at the highest concentration of ACTH for the longest time (20 mg/g-0.52 mg/cm
2/t
1/2) (
Table 6). The Higuchi model is based on the hypotheses that the initial drug concentration is much higher than the drug solubility, matrix swelling and dissolution are negligible, drug diffusivity is constant, and perfect sink conditions are attained in the release environment [
44]. The dependence of the release rate on the ACTH concentration was observed. The higher the concentration, the longer the dissolution and therefore the release time. The release results may be influenced by the nature of the ointment medium, which enables the diffusion process, because in previous studies it was shown that there is no diffusion from such bases of Eucerine or Lekobaza Lux. Among ointment formulations with three different concentrations (15, 20, and 25 mg/g), the ointment with the lowest concentration of ACTH (15 mg/g) in the release test turned out to be the most advantageous, as ACTH was released from this formulation the fastest. The longest ACTH was released from the ointment at a concentration of 25 mg/g and this preparation can provide the effect of prolonged hormone release from the ointment. As far as rheological properties are concerned, the ointment with a concentration of 15 mg/g, from which ACTH was released the fastest, was also characterized by the lowest viscosity in the range of 100–1000 s
−1, as shown in
Table 3 and
Figure 4.
The literature reports show that the release process depends on the properties of the membrane through which the therapeutic substance diffuses into the acceptor fluid. It has been reported that artificial cellulose membranes give more reproducible results than natural pork membranes due to the way the natural membrane is prepared. Due to their lipophilic nature, porcine membranes may be more permeable to some substances, whereas artificial ones may be more beneficial to other substances [
45,
46,
47,
48,
49]. In turn, the use of hairless mouse skin leads to overstated results [
50]. The work on synthetic membranes for percutaneous and topical supplies focused on the use of polymeric materials, usually based on silicone. Such membranes are ideal for ex vivo skin replacement as they can be prepared with a specific thickness, are easy to handle and store, are relatively cheap, neutral, and provide reproducible results [
51].
The release of ACTH was a preliminary study; therefore, regenerated cellulose membranes were used to optimize the results. Based on the test of release through artificial membranes, it can be preliminarily estimated whether it is possible at all to penetrate large 4.5 kDa particles of ACTH through the skin. In the human skin penetration test, the release process will certainly be slightly different, and this effect can be enhanced by using absorption promoters. The problem of polypeptide permeation through human skin is the next stage of our research.
There are no reports on the penetration of ACTH through the skin in the literature. The idea of administering ACTH as an ointment is a new project, so if you formulate a new form of the drug, it starts with the simplest possible recipe, which can always be improved in the next stages of research development.
The diffusion of a drug molecule depends on many factors: The character of the molecule, its size, drug form, water solubility, oil/water partition coefficient, and physicochemical properties of the drug form. Ideally, when the molecular weight does not exceed 3 kDa or even below 500 Da, log P 1–3, but above 4 are also absorbed [
4]. Semi-solid preparations in the form of ointment containing substances, such as heparin, with a particle size from 3000 to 30,000 g/mol, insulin 5809 g/mol, oestradiol—272.38 g/mol, ethinylestradiol—296.40 g/mol, and human gonadotropin—36–46 kDa, are used in the treatment. It is more beneficial when the substance is molecular rather than ionic, as when it is acidic or alkaline rather than saline, lipophilic molecules penetrate skin structures better than hydrophilic ones. However, different forms are used to increase the penetration of molecules through the skin layers, e.g., different absorption promoters can be added, including urea, dimethylsulphoxide (DMSO), and albumin, which modify the release of the therapeutic substance from the dosage form. It is also possible to modify the composition of the ointment medium in order to better release the hormone or the technique of introducing of the active substance into the vehicle [
4,
52].