madman
Super Moderator
ABSTRACT
Cutaneous permeation assays are crucial to attest to the performance or bioequivalence of topical or transdermal products. Although the official guidelines (e.g., FDA/EMA) play a key role in harmonizing the experimental design, alternative methods are often proposed by the scientific community, which makes it difficult to compare results from different studies. In this review, permeation assays with testosterone (TST) were selected to show this high variability in drug transport rate. The main sources of variation discussed were tissue thickness, animal model, donor, and receptor fluid constitution, type of solubilizing agent used in aqueous fluids, drug concentration, degree of supersaturation, skin lipid content, number of experimental times, and the physical-chemical stability of the molecule in test fluids. This variation becomes even more critical for molecules that present biopharmaceutical limitations such as TST. In addition, the skin presents specific receptors for this hormone due to its physiological action in this region of the body, which makes the evaluation of the TST transport rate in this tissue even more challenging. The impact of each experimental parameter mentioned above on the flux or permeation coefficient of TST is discussed in detail in the review. Assays used to evaluate tissue integrity are also presented.
1. INTRODUCTION
Many topically administrated therapeutic agents present a limited or suboptimal efficacy due to low penetration into the skin. Solubility and partition coefficient are key aspects related to drug transport through the skin. Overall, high solubility results in high drug concentration in the donor phase (or region of application of the formulation), improving the permeation flux. The drug partitioning from the donor phase into skin layers, in turn, represents the rate-limiting step for drug flux when these molecules are characterized by a poor aqueous solubility taking into account the lipid constitution of biological membranes (Ceschel et al., 2005).
Steroidal hormones, for example, are characterized by a low aqueous solubility (Norman and Litwack, 1997), requiring the investigation of technological/formulation approaches aiming to increase the free drug concentration in the donor phase. Drug complexation (e.g. association with cyclodextrins), incorporation in vesicular or particulate systems (e.g. micro-and nanoparticles), and supersaturated systems have been often considered for this purpose (Ceschel et al., 2005).
On the other hand, the low aqueous solubility of hormones allows drug partitioning from the donor phase into skin layers. Due to the high value of log P, these compounds bind so strongly to the tissues, particularly the more superficial layers (SC and viable epidermis), resulting in a RESERVOIR EFFECT and LOW TRANSPORT RATE to the dermis (Magnusson et al., 2006). This event is particularly noticed for TST after the application of low concentrations of this agent on the skin (Schlupp et al., 2014).
In addition to the slow tissue diffusion because of the high affinity by lipids and high molar mass, the skin presents receptors for these compounds due to the physiological action they play in that region of the body. The TST interacts directly with androgen receptors (ARs), which have been localized in most epidermis keratinocytes. In the dermis, ARs were found in approximately 10% of fibroblasts. AR expression was also found in both basal cells and sebocytes of sebaceous glands whereas it was restricted to dermal papilla cells in hair follicles (Pelletier and Ren, 2004). A modest reduction in epidermal thickness is also observed in TST replacement therapies (Kao et al., 2001). In view of this high affinity of sex hormones by skin tissues, the inclusion of chemical absorption enhancer combinations able of providing membrane fluidification or lipid extraction is often recommended during the development of novel formulations. These compounds may interact with both lipid and polar domains (Magnusson et al., 2006) and thus chemical absorption enhancers of both polarities should be considered.
The evaluation of percutaneous permeation of molecules is a key step when new dermal or transdermal delivery systems are purposed and different in silico, ex vivo, and in vivo models may be considered. Although in vivo human assays represent the gold standard, ethical, practical, or economic reasons have limited their use. Alternatively, tissues from different animal models, particularly pig ear skin, have been selected in many permeation studies. These tissues are easier to obtain compared to human tissues, however, high experimental variability may be found. Barrier properties may also vary from human skin depending on the selected animal model (Abd et al., 2016).
In addition to the inherent variability of the special animal considered, the tissue thickness and preparation method, the composition of the receptor, and donor fluid can also represent sources of experimental variation. All these aspects will be discussed detailly in this review article, which considered the TST as a drug model. This drug was selected based on its biopharmaceutical limitations (low solubility and reservoir effect), the various permeation studies already carried out with this compound, and its wide use in hormone replacement therapies. Although TST therapy has effectively treated hypogonadism for decades, therapies simpler and more convenient to use, safer, and able to mimic physiological levels are still needed (Kaminetsky and Wynia, 2015). The main objective of this study is to provide tools so that future permeation assays can be planned more rationally considering a series of problems that may be found in each step assay. Once free drug solutions are frequently used as the control during the evaluation of the performance of new topical formulations in skin permeation studies, this review focused on the analysis of studies with free TST solutions/suspensions. The solubility of this molecule in different solvents, the importance of this physicochemical parameter in the design of permeation studies as well as aspects related to the drug quantification steps are described in detail.
2. SOLUBILITY STUDIES
3. RECEPTOR PHASE SELECTION
4. EFFECT OF DRUG CONCENTRATION FROM DONOR PHASE
5. IMPACT OF TISSUE PREPARATION ON DRUG PERMEATION
6. SELECTION OF ANIMAL MODELS OR MEMBRANE TYPE
7. ASSAY TOTAL DURATION & SAMPLING INTERVALS
8. SKIN INTEGRITY ASSAYS
9. SAMPLE STORAGE BEFORE QUANTIFICATION
10. DRUG QUANTIFICATION STEP
11. DRUG RETENTION
12. FINAL CONSIDERATIONS
In summary, this review contributes to defining more properly the experimental variables of cutaneous permeation assays with poorly soluble molecules such as the TST, creating new discussions for future revisions of official guidelines.
Cutaneous permeation assays are crucial to attest to the performance or bioequivalence of topical or transdermal products. Although the official guidelines (e.g., FDA/EMA) play a key role in harmonizing the experimental design, alternative methods are often proposed by the scientific community, which makes it difficult to compare results from different studies. In this review, permeation assays with testosterone (TST) were selected to show this high variability in drug transport rate. The main sources of variation discussed were tissue thickness, animal model, donor, and receptor fluid constitution, type of solubilizing agent used in aqueous fluids, drug concentration, degree of supersaturation, skin lipid content, number of experimental times, and the physical-chemical stability of the molecule in test fluids. This variation becomes even more critical for molecules that present biopharmaceutical limitations such as TST. In addition, the skin presents specific receptors for this hormone due to its physiological action in this region of the body, which makes the evaluation of the TST transport rate in this tissue even more challenging. The impact of each experimental parameter mentioned above on the flux or permeation coefficient of TST is discussed in detail in the review. Assays used to evaluate tissue integrity are also presented.
1. INTRODUCTION
Many topically administrated therapeutic agents present a limited or suboptimal efficacy due to low penetration into the skin. Solubility and partition coefficient are key aspects related to drug transport through the skin. Overall, high solubility results in high drug concentration in the donor phase (or region of application of the formulation), improving the permeation flux. The drug partitioning from the donor phase into skin layers, in turn, represents the rate-limiting step for drug flux when these molecules are characterized by a poor aqueous solubility taking into account the lipid constitution of biological membranes (Ceschel et al., 2005).
Steroidal hormones, for example, are characterized by a low aqueous solubility (Norman and Litwack, 1997), requiring the investigation of technological/formulation approaches aiming to increase the free drug concentration in the donor phase. Drug complexation (e.g. association with cyclodextrins), incorporation in vesicular or particulate systems (e.g. micro-and nanoparticles), and supersaturated systems have been often considered for this purpose (Ceschel et al., 2005).
On the other hand, the low aqueous solubility of hormones allows drug partitioning from the donor phase into skin layers. Due to the high value of log P, these compounds bind so strongly to the tissues, particularly the more superficial layers (SC and viable epidermis), resulting in a RESERVOIR EFFECT and LOW TRANSPORT RATE to the dermis (Magnusson et al., 2006). This event is particularly noticed for TST after the application of low concentrations of this agent on the skin (Schlupp et al., 2014).
In addition to the slow tissue diffusion because of the high affinity by lipids and high molar mass, the skin presents receptors for these compounds due to the physiological action they play in that region of the body. The TST interacts directly with androgen receptors (ARs), which have been localized in most epidermis keratinocytes. In the dermis, ARs were found in approximately 10% of fibroblasts. AR expression was also found in both basal cells and sebocytes of sebaceous glands whereas it was restricted to dermal papilla cells in hair follicles (Pelletier and Ren, 2004). A modest reduction in epidermal thickness is also observed in TST replacement therapies (Kao et al., 2001). In view of this high affinity of sex hormones by skin tissues, the inclusion of chemical absorption enhancer combinations able of providing membrane fluidification or lipid extraction is often recommended during the development of novel formulations. These compounds may interact with both lipid and polar domains (Magnusson et al., 2006) and thus chemical absorption enhancers of both polarities should be considered.
The evaluation of percutaneous permeation of molecules is a key step when new dermal or transdermal delivery systems are purposed and different in silico, ex vivo, and in vivo models may be considered. Although in vivo human assays represent the gold standard, ethical, practical, or economic reasons have limited their use. Alternatively, tissues from different animal models, particularly pig ear skin, have been selected in many permeation studies. These tissues are easier to obtain compared to human tissues, however, high experimental variability may be found. Barrier properties may also vary from human skin depending on the selected animal model (Abd et al., 2016).
In addition to the inherent variability of the special animal considered, the tissue thickness and preparation method, the composition of the receptor, and donor fluid can also represent sources of experimental variation. All these aspects will be discussed detailly in this review article, which considered the TST as a drug model. This drug was selected based on its biopharmaceutical limitations (low solubility and reservoir effect), the various permeation studies already carried out with this compound, and its wide use in hormone replacement therapies. Although TST therapy has effectively treated hypogonadism for decades, therapies simpler and more convenient to use, safer, and able to mimic physiological levels are still needed (Kaminetsky and Wynia, 2015). The main objective of this study is to provide tools so that future permeation assays can be planned more rationally considering a series of problems that may be found in each step assay. Once free drug solutions are frequently used as the control during the evaluation of the performance of new topical formulations in skin permeation studies, this review focused on the analysis of studies with free TST solutions/suspensions. The solubility of this molecule in different solvents, the importance of this physicochemical parameter in the design of permeation studies as well as aspects related to the drug quantification steps are described in detail.
2. SOLUBILITY STUDIES
3. RECEPTOR PHASE SELECTION
4. EFFECT OF DRUG CONCENTRATION FROM DONOR PHASE
5. IMPACT OF TISSUE PREPARATION ON DRUG PERMEATION
6. SELECTION OF ANIMAL MODELS OR MEMBRANE TYPE
7. ASSAY TOTAL DURATION & SAMPLING INTERVALS
8. SKIN INTEGRITY ASSAYS
9. SAMPLE STORAGE BEFORE QUANTIFICATION
10. DRUG QUANTIFICATION STEP
11. DRUG RETENTION
12. FINAL CONSIDERATIONS
In summary, this review contributes to defining more properly the experimental variables of cutaneous permeation assays with poorly soluble molecules such as the TST, creating new discussions for future revisions of official guidelines.
